PCR
Long Distance PCR
PCR Primers
UV Irradiation for De-Contamination
RT-PCR
Quantitative RT-PCR
Semi-Quantitative RT-PCR: Competitive RT-PCR
Semi-Quantitative RT-PCR: Noncompetitive RT-PCR
In situ PCR
In situ RT-PCR
PCR in situ Hybridization
Sunday, September 21, 2008
Sunday, September 14, 2008
History of Polymerase chain reaction (PCR)--(1)
From Wikipedia, the free encyclopedia
The history of the Polymerase Chain Reaction (or PCR) has variously been described as a classic "Eureka!" moment[1], or as an example of cooperative teamwork between disparate researchers[2]. A list of some of the events before, during, and after its development:
Prelude
On April 25, 1953 James D. Watson and Francis Crick publish "a radically different structure" for DNA[3], thereby founding the field of Molecular Genetics. Their structure involves two strands of complementary base-paired DNA, running in opposite directions as a double helix. They conclude their report saying that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material". They are awarded the Nobel Prize in 1962.
Starting in the mid 1950s, Arthur Kornberg begins to study the mechanism of DNA replication[4]. By 1957 he has identified the first DNA polymerase[5]. The enzyme is surprisingly limited, creating DNA in just one direction and requiring an existing primer to initiate copying of the template strand. However, the overall DNA replication process is surprisingly complex, requiring separate proteins to open the DNA helix, to keep it open, to create primers, to synthesize new DNA, to remove the primers, and to tie the pieces all together. He is awarded the Nobel Prize in 1959.
In the early 1960s H. Gobind Khorana participates in the discovery of the Genetic Code. Afterwards, he initiates a large project to totally synthesize a functional human gene[6]. To achieve this, he pioneers many of the techniques needed to make and use synthetic DNA oligonucleotides. Sequence-specific oligos are used both as building blocks for the gene, and as primers and templates for DNA polymerase. In 1968 Khorana is awarded the Nobel Prize for his work on the Genetic Code.
In 1969 Thomas Brock reports the isolation of a new species of bacterium from a hot spring in Yellowstone National Park. Naming it Thermus aquaticus[7] (Taq), it goes on to become a standard source of enzymes able to withstand higher temperatures than those from E. Coli.
In 1970 a modified version of DNA Polymerase I from E. coli is reported[8]. Treatment with a protease removes the 'forward' nuclease activity of this enzyme. The overall activity of the resulting Klenow fragment is therefore biased towards the synthesis of DNA, rather than its degradation.
By 1971 researchers in Khorana's project, concerned over their yields of DNA, begin looking at "repair synthesis" - an artificial system of primers and templates that allows DNA polymerase to copy segments of the gene they are synthesizing. Although similar to PCR in using repeated applications of DNA polymerase, the process they usually describe[9] employs just a single primer-template complex, and therefore would not lead to the exponential amplification seen in PCR.
Also by 1971 Kjell Kleppe, a researcher in Khorana's lab, envisions a process very similar to PCR. At the end of a paper on the earlier technique[10], he describes how a two-primer system might lead to replication of a specific segment of DNA:
"... one would hope to obtain two structures, each containing the full length of the template strand appropriately complexed
with the primer. DNA polymerase will be added to complete the process of repair replication. Two molecules of the original
duplex should result. The whole cycle could be repeated, there being added every time a fresh dose of the enzyme." [10]
No results are shown there, and the mention of unpublished experiments in another paper[9] may (or may not) refer to the two-primer replication system. (These early precursors to PCR were carefully scrutinized in a patent lawsuit, and are discussed in Mullis' chapters in [11].)
Also in 1971, Cetus Corporation is founded in Berkeley, California by Ronald Cape, Peter Farley, and Donald Glaser. Initially the company screens for microorganisms capable of producing components used in the manufacture of food, chemicals, vaccines, or pharmaceuticals. After moving to nearby Emeryville, they take up projects involving the new biotechnology industry, primarily the cloning and expression of human genes, but also the development of diagnostic tests for genetic mutations.
In 1976 a DNA polymerase[12] is isolated from T. aquaticus. It is found to retain its activity at temperatures above 75°C.
In 1977 Frederick Sanger reports a method for determining the sequence of DNA[13]. The technique involves an oligonucleotide primer, DNA polymerase, and modified nucleotide precursors that block further extension of the primer in sequence-dependent manner. He is awarded the Nobel Prize in 1980.
Thus, by 1980 all of the components needed to perform PCR amplification were known to the scientific community. The use of DNA polymerase to extend oligonucleotide primers was a common procedure in DNA sequencing and the production of cDNA for cloning and expression. The use of DNA polymerase for nick translation was the most common method used to label DNA probes for Southern blotting.
Theme
In 1979 Cetus Corporation hires Kary Mullis to synthesize oligonucleotides for various research and development projects throughout the company[14]. These oligos are used as probes for screening cloned genes, as primers for DNA sequencing and cDNA synthesis, and as building blocks for gene construction. Originally synthesizing these oligos by hand, Mullis later evaluates early prototypes for automated synthesizers[1].
By May 1983 Mullis has synthesized oligo probes for a project at Cetus attempting to analyze a mutation for a human genetic disease. Hearing of problems with their work, Mullis envisions an alternative technique based on Sanger's DNA sequencing method[14]. Realizing the difficulty in making that method specific to a single location in the genome, Mullis considers adding a second primer on the opposite strand. He then generalizes the idea, and realizes that repeated applications of polymerase could lead to a chain reaction of replication for a specific segment of the genome - PCR.
Later in 1983 Mullis begins to test his idea. His first experiment[2] does not involve thermal cycling - he hopes that the polymerase can perform continued replication on its own. Later experiments that year do involve repeated thermal cycling, and target small segments of a cloned gene. Mullis considers these experiments a success, but is unable to convince other researchers.
In June 1984 Cetus holds its annual meeting in Monterey, California. Its scientists and consultants present their results, and consider future projects. Mullis presents a poster on the production of oligonucleotides by his laboratory, and shows some of the results from his experiments with PCR[2]. Only Joshua Lederberg, a Cetus consultant, shows any interest[14]. Later at the meeting, Mullis is involved in a physical altercation with another Cetus researcher, over a dispute unrelated to PCR[2]. The other scientist soon leaves the company, and Mullis is removed as head of the oligo synthesis lab. The days of his continued employment at Cetus may be numbered.
Development
In September of 1984 Tom White, VP of Research at Cetus (and a close friend), pressures Mullis to take his idea to the group developing the genetic mutation assay. Together, they spend the following months designing experiments that could convincingly show that PCR is working on genomic DNA. Unfortunately, the expected amplification product is not visible in agarose gel electrophoresis[15], leading to confusion as to whether the reaction has any specificity to the targeted region.
In November of 1984[2] the amplification products are analyzed by Southern blotting, which clearly shows an increasing amount of the expected 110 bp DNA product[16]. Having the first visible signal, the researchers are able to begin finding optimum conditions for the reaction. Later, the amplified products are cloned and sequenced, showing that only a small fraction of the amplified DNA is the desired target, and that the polymerase then being used only rarely incorporates incorrect nucleotides during replication[15].
The history of the Polymerase Chain Reaction (or PCR) has variously been described as a classic "Eureka!" moment[1], or as an example of cooperative teamwork between disparate researchers[2]. A list of some of the events before, during, and after its development:
Prelude
On April 25, 1953 James D. Watson and Francis Crick publish "a radically different structure" for DNA[3], thereby founding the field of Molecular Genetics. Their structure involves two strands of complementary base-paired DNA, running in opposite directions as a double helix. They conclude their report saying that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material". They are awarded the Nobel Prize in 1962.
Starting in the mid 1950s, Arthur Kornberg begins to study the mechanism of DNA replication[4]. By 1957 he has identified the first DNA polymerase[5]. The enzyme is surprisingly limited, creating DNA in just one direction and requiring an existing primer to initiate copying of the template strand. However, the overall DNA replication process is surprisingly complex, requiring separate proteins to open the DNA helix, to keep it open, to create primers, to synthesize new DNA, to remove the primers, and to tie the pieces all together. He is awarded the Nobel Prize in 1959.
In the early 1960s H. Gobind Khorana participates in the discovery of the Genetic Code. Afterwards, he initiates a large project to totally synthesize a functional human gene[6]. To achieve this, he pioneers many of the techniques needed to make and use synthetic DNA oligonucleotides. Sequence-specific oligos are used both as building blocks for the gene, and as primers and templates for DNA polymerase. In 1968 Khorana is awarded the Nobel Prize for his work on the Genetic Code.
In 1969 Thomas Brock reports the isolation of a new species of bacterium from a hot spring in Yellowstone National Park. Naming it Thermus aquaticus[7] (Taq), it goes on to become a standard source of enzymes able to withstand higher temperatures than those from E. Coli.
In 1970 a modified version of DNA Polymerase I from E. coli is reported[8]. Treatment with a protease removes the 'forward' nuclease activity of this enzyme. The overall activity of the resulting Klenow fragment is therefore biased towards the synthesis of DNA, rather than its degradation.
By 1971 researchers in Khorana's project, concerned over their yields of DNA, begin looking at "repair synthesis" - an artificial system of primers and templates that allows DNA polymerase to copy segments of the gene they are synthesizing. Although similar to PCR in using repeated applications of DNA polymerase, the process they usually describe[9] employs just a single primer-template complex, and therefore would not lead to the exponential amplification seen in PCR.
Also by 1971 Kjell Kleppe, a researcher in Khorana's lab, envisions a process very similar to PCR. At the end of a paper on the earlier technique[10], he describes how a two-primer system might lead to replication of a specific segment of DNA:
"... one would hope to obtain two structures, each containing the full length of the template strand appropriately complexed
with the primer. DNA polymerase will be added to complete the process of repair replication. Two molecules of the original
duplex should result. The whole cycle could be repeated, there being added every time a fresh dose of the enzyme." [10]
No results are shown there, and the mention of unpublished experiments in another paper[9] may (or may not) refer to the two-primer replication system. (These early precursors to PCR were carefully scrutinized in a patent lawsuit, and are discussed in Mullis' chapters in [11].)
Also in 1971, Cetus Corporation is founded in Berkeley, California by Ronald Cape, Peter Farley, and Donald Glaser. Initially the company screens for microorganisms capable of producing components used in the manufacture of food, chemicals, vaccines, or pharmaceuticals. After moving to nearby Emeryville, they take up projects involving the new biotechnology industry, primarily the cloning and expression of human genes, but also the development of diagnostic tests for genetic mutations.
In 1976 a DNA polymerase[12] is isolated from T. aquaticus. It is found to retain its activity at temperatures above 75°C.
In 1977 Frederick Sanger reports a method for determining the sequence of DNA[13]. The technique involves an oligonucleotide primer, DNA polymerase, and modified nucleotide precursors that block further extension of the primer in sequence-dependent manner. He is awarded the Nobel Prize in 1980.
Thus, by 1980 all of the components needed to perform PCR amplification were known to the scientific community. The use of DNA polymerase to extend oligonucleotide primers was a common procedure in DNA sequencing and the production of cDNA for cloning and expression. The use of DNA polymerase for nick translation was the most common method used to label DNA probes for Southern blotting.
Theme
In 1979 Cetus Corporation hires Kary Mullis to synthesize oligonucleotides for various research and development projects throughout the company[14]. These oligos are used as probes for screening cloned genes, as primers for DNA sequencing and cDNA synthesis, and as building blocks for gene construction. Originally synthesizing these oligos by hand, Mullis later evaluates early prototypes for automated synthesizers[1].
By May 1983 Mullis has synthesized oligo probes for a project at Cetus attempting to analyze a mutation for a human genetic disease. Hearing of problems with their work, Mullis envisions an alternative technique based on Sanger's DNA sequencing method[14]. Realizing the difficulty in making that method specific to a single location in the genome, Mullis considers adding a second primer on the opposite strand. He then generalizes the idea, and realizes that repeated applications of polymerase could lead to a chain reaction of replication for a specific segment of the genome - PCR.
Later in 1983 Mullis begins to test his idea. His first experiment[2] does not involve thermal cycling - he hopes that the polymerase can perform continued replication on its own. Later experiments that year do involve repeated thermal cycling, and target small segments of a cloned gene. Mullis considers these experiments a success, but is unable to convince other researchers.
In June 1984 Cetus holds its annual meeting in Monterey, California. Its scientists and consultants present their results, and consider future projects. Mullis presents a poster on the production of oligonucleotides by his laboratory, and shows some of the results from his experiments with PCR[2]. Only Joshua Lederberg, a Cetus consultant, shows any interest[14]. Later at the meeting, Mullis is involved in a physical altercation with another Cetus researcher, over a dispute unrelated to PCR[2]. The other scientist soon leaves the company, and Mullis is removed as head of the oligo synthesis lab. The days of his continued employment at Cetus may be numbered.
Development
In September of 1984 Tom White, VP of Research at Cetus (and a close friend), pressures Mullis to take his idea to the group developing the genetic mutation assay. Together, they spend the following months designing experiments that could convincingly show that PCR is working on genomic DNA. Unfortunately, the expected amplification product is not visible in agarose gel electrophoresis[15], leading to confusion as to whether the reaction has any specificity to the targeted region.
In November of 1984[2] the amplification products are analyzed by Southern blotting, which clearly shows an increasing amount of the expected 110 bp DNA product[16]. Having the first visible signal, the researchers are able to begin finding optimum conditions for the reaction. Later, the amplified products are cloned and sequenced, showing that only a small fraction of the amplified DNA is the desired target, and that the polymerase then being used only rarely incorporates incorrect nucleotides during replication[15].
History of Polymerase chain reaction (PCR)--(2)
Exposition
As per normal industrial practice, the results are first used to apply for patents. Mullis prepares an application[17] for the basic idea of PCR and many potential applications, and is asked by the PTO to include more results. On March 28, 1985 the entire development group (including Mullis) files an application[18] that is more focused on the analysis of the SCA mutation via PCR and OR. After modification, both patents are approved on July 28, 1987.
In the spring of 1985 the development group begins to apply PCR to other targets. Primers and probes are designed for a variable segment of the HLA DQα gene. This reaction turns out to be much more specific than that for the β-hemoglobin target - the expected PCR product[15] is directly visible on agarose gel electrophoresis. The amplification products from various sources are also cloned and sequenced, the first determination of new alleles by PCR[15]. At this same time the original OR assay technique is replaced with the more general ASO method[19].
Also early in 1985, the group turns its attention to the use of a thermostable DNA polymerase (the enzyme used in the original reaction is destroyed at each heating step). A literature search[1] reveals that only two have been described, from Taq and Bst. The report on Taq polymerase[12] is more detailed, so it is chosen for testing. A fortuitous decision - the Bst polymerase is later found to be unsuitable for PCR[citation needed]. That summer Mullis tries twice to isolate the enzyme, and a group outside of Cetus is also contracted to make it, all without success. In the Fall of 1985 Susanne Stoffel and David Gelfand at Cetus succeed in making the polymerase, and it is immediately found by Randy Saiki to support the PCR process.
With patents submitted, work proceeds for reporting PCR to the general scientific community. An abstract for a meeting in Salt Lake City is submitted in April 1985, and the first announcement of PCR is made there by Saiki in October[20]. Two publications are planned - an 'idea' paper from Mullis, and an 'application' paper from the entire development group. Mullis submits his manuscript to the journal Nature, which rejects it for not including results. The other paper, mainly describing the OR analysis assay, is submitted to Science on September 20, 1985 and is accepted in November. After the rejection of Mullis' report in December, details on the PCR process are hastily added to the second paper, which appears on December 20, 1985[16].
In May of 1986 Mullis presents PCR at the Cold Spring Harbor Symposium[21], and publishes a modified version of his original 'idea' manuscript much later[22]. The first non-Cetus report using PCR is submitted on September 5, 1986[23], indicating how quickly other laboratories are implementing the technique. The Cetus development group publishes their detailed sequence analysis of PCR products on September 8, 1986[15], and their use of ASO probes on November 13, 1986[19].
The use of Taq polymerase in PCR is announced by Henry Erlich at a meeting in Berlin on September 20, 1986, is submitted for publication in October of 1987, and is published early the next year'[24]. The patent for PCR with Taq polymerase is filed on June 17, 1987, and is issued on October 23, 1990[25].
Variation
In December 1985 a joint venture between Cetus and Perkin-Elmer is established to develop instruments and reagents for PCR. Complex Thermal Cyclers are constructed to perform the Klenow-based amplifications, but are never marketed. Simpler machines for Taq-based PCR are developed, and on November 19, 1987 a press release announces the commercial availability of the "PCR-1000 Thermal Cycler" and "AmpliTaq DNA Polymerase".
In the Spring of 1985 John Sninsky at Cetus begins to apply PCR to the difficult task of quantitating the amount of HIV circulating in blood. A viable test is announced on April 11, 1986, and is published in May 1987[26] . Donated blood can now be screened for the virus, and the effect of antiviral drugs can be directly monitored.
In 1985 Norm Arnheim, also a member of the development team, concludes his sabbatical at Cetus and gets a real job at USC. He begins to investigate the use of PCR to amplifiy samples containing just a single copy of the target sequence. By 1989 his lab runs mutiplex-PCR on single sperm to directly analyze the products of meiotic recombination[27]. These single-copy amplifications, which had first been run during the characterization of Taq polymerase[24], become vital to the study of ancient DNA, as well as the genetic typing of preimplanted embryos.
In 1986 Edward Blake, a forensics scientist working in the Cetus building, collaborates with Bruce Budowle (of the FBI) and Cetus researchers to apply PCR to the analysis of criminal evidence. A panel of DNA samples from old cases is collected and coded, and is analyzed blind by Saiki using the HLA DQα assay. When the code is broken, all of the evidence and perpetrators match. Blake uses the technique almost immediately in "Pennsylvania v. Pestinikas"[28], the first use of PCR in a criminal case. This DQα test is developed by Cetus as one of their "Ampli-Type" kits, and goes on to become part of early protocols for the testing of forensic evidence.
By 1989 Alec Jeffreys, who had earlier developed and applied the first DNA Fingerprinting tests, uses PCR to increase their sensitivity[29]. With further modification, the amplification of highly polymorphic VNTR loci will become the standard protocol for National DNA Databases such as CODIS. The guilty go to jail, and the ability of PCR to restest old evidence begins to set the innocent free.
In 1987 Russ Higuchi succeeds in amplifying DNA from a human hair[30]. This work expands to develop methods to amplify DNA from highly degraded samples, such as from Ancient DNA and in forensic evidence. On January 30, 1989 an episode of Star Trek: The Next Generation airs. The ship's doctor is being rapidly aged by a virus attacking her DNA, and is cured when her pre-infection DNA is isolated from a hair found in her cabin. PCR has entered the mainstream media.
Coda
On December 22, 1989 the journal Science awards Taq Polymerase (and PCR) its first "Molecule of the Year". The 'Taq PCR' paper[24] goes on to become (for several years) the most cited publication in biology.
After the publication of the first PCR paper[16], the United States Government sends a stern letter to Randy Saiki, admonishing him for publishing a report on "chain reactions" without the required prior review and approval by the U.S. Department of Energy. Cetus writes back, explaining the differences between PCR and the atomic bomb.
On July 23, 1991 Cetus announces that it will be sold to its neighboring biotechnology company Chiron. As part of the sale, rights to the PCR patents are sold for USD $300 million to Hoffman-La Roche (who in 1989 had bought limited rights to PCR). Many of the Cetus PCR researchers move to a new subsidiary, Roche Molecular Systems.
On October 13, 1993 Kary Mullis, who had left Cetus in 1986, is awarded the Nobel Prize in Chemistry. On the morning of his acceptance speech[1], he is nearly arrested by Swedish authorities for the "inappropriate use of a laser pointer"[31].
References
^ a b c d Kary Mullis' Nobel Lecture, December 8, 1993
^ a b c d e Rabinow P "Making PCR: A Story of Biotechnology" University of Chicago Press (1996) ISBN 0-226-70147-6
^ Watson JD, Crick FHC "A Structure for Deoxyribose Nucleic Acid", Nature vol. 171, pp. 737-738 (1953). [1]
^ (Arthur Kornberg's Discovery of DNA Polymerase I) J. Biol. Chem. vol. 280, p. 46. [2]
^ Lehman, IR, Bessman MJ, Simms ES, Kornberg A "Enzymatic Synthesis of Deoxyribonucleic Acid. I. Preparation of Substrates and Partial Purification of an Enzyme from Escherichia coli" J. Biol. Chem. vol. 233(1) pp. 163-170 (1958).
^ Khorana HG et al. "Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 1. General introduction" J. Biol. Chem. vol. 251(3) pp. 565-70 (1976).
^ Brock TD, Freeze H "Thermus aquaticus, a Nonsporulating Extreme Thermophile" J. Bact. vol. 98(1) pp. 289-297 (1969).
^ Klenow H and Henningsen I "Selective Elimination of the Exonuclease Activity of the Deoxyribonucleic Acid Polymerase from Escherichia coli B by Limited Proteolysis" Proc Natl Acad Sci vol. 65 pp. 168-75 (1970).
^ a b Panet A, Khorana HG "Studies on Polynucleotides" J. Biol. Chem. vol. 249(16), pp. 5213-21 (1974).
^ a b Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG "Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases." J. Molec. Biol. vol. 56, pp. 341-61 (1971).
^ Mullis KB, Ferré F, Gibbs RA "The Polymerase Chain Reaction" Birkhäuser Press (1994) ISBN 0-817-63750-8
^ a b Chien A, Edgar DB, Trela JM "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus" J. Bact. vol. 174 pp. 1550-1557 (1976).
^ Sanger F, Nicklen S, Coulson AR "DNA sequencing with chain-terminating inhibitors" Proc Natl Acad Sci vol. 74(12) pp. 5463-7 (1977).
^ a b c Mullis KB "The Unusual Origins of the Polymerase Chain Reaction" Scientific American, vol. 262, pp. 56-65 (April 1990).
^ a b c d e Scharf et al. "Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences" Science vol. 233, pp. 1076-78 (1986).
^ a b c Saiki RK et al. "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia" Science vol. 230 pp. 1350-54 (1985).
^ Mullis KB "Process for amplifying nucleic acid sequences." U.S. Patent 4,683,202.
^ Mullis, KB et al. "Process for amplifying, detecting, and/or-cloning nucleic acid sequences." U.S. Patent 4,683,195.
^ a b Saiki et al. "Analysis of enzymatically amplified β-globin and HLA DQα DNA with allele-specific oligonucleotide probes." Nature vol. 324 (6093) pp. 163-6 (1986).
^ Saiki, R et al. "A Novel Method for the Prenatal Diagnosis of Sickle Cell Anemia" Amer. Soc. Human Genetics, Oct. 9-13, 1985.
^ Mullis KB et al. "Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction." Cold Spring Harbor Symp. Quant. Biol. vol. 51 pp. 263-73 (1986).
^ Mullis KB and Faloona FA "Specific Synthesis of DNA in vitro via a Polymerase-Catalyzed Chain Reaction." Methods in Enzymology vol. 155(F) pp. 335-50 (1987).
^ Verlaan-de Vries M et al. "A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides." Gene vol. 50(1-3) pp. 313-20 (1986).
^ a b c Saiki et al. "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science vol. 239 pp. 487-91 (1988).
^ Mullis, KB et al. "Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme." U.S. Patent 4,965,188.
^ Kwok S et al. "Identification of HIV sequences by using in vitro enzymatic amplification and oligomer cleavage detection." J. Virol. vol. 61(5) pp. 1690-4 (1987).
^ Boehnke M et al. "Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm." Am J Hum Genet vol. 45(1) pp. 21-32 (1989).
^ Forensic Science Timeline (PDF).
^ Jeffreys A et al. "Amplification of human minisatellites." Nucleic Acids Research vol. 23 pp. 10953-71 (1988).
^ Higuchi R et al. "DNA typing from single hairs." Nature vol. 332(6164) pp. 543-6 (1988).
^ Mullis KB "Dancing Naked in the Mind Field" Pantheon Books (1998) ISBN 0-679-44255-3
As per normal industrial practice, the results are first used to apply for patents. Mullis prepares an application[17] for the basic idea of PCR and many potential applications, and is asked by the PTO to include more results. On March 28, 1985 the entire development group (including Mullis) files an application[18] that is more focused on the analysis of the SCA mutation via PCR and OR. After modification, both patents are approved on July 28, 1987.
In the spring of 1985 the development group begins to apply PCR to other targets. Primers and probes are designed for a variable segment of the HLA DQα gene. This reaction turns out to be much more specific than that for the β-hemoglobin target - the expected PCR product[15] is directly visible on agarose gel electrophoresis. The amplification products from various sources are also cloned and sequenced, the first determination of new alleles by PCR[15]. At this same time the original OR assay technique is replaced with the more general ASO method[19].
Also early in 1985, the group turns its attention to the use of a thermostable DNA polymerase (the enzyme used in the original reaction is destroyed at each heating step). A literature search[1] reveals that only two have been described, from Taq and Bst. The report on Taq polymerase[12] is more detailed, so it is chosen for testing. A fortuitous decision - the Bst polymerase is later found to be unsuitable for PCR[citation needed]. That summer Mullis tries twice to isolate the enzyme, and a group outside of Cetus is also contracted to make it, all without success. In the Fall of 1985 Susanne Stoffel and David Gelfand at Cetus succeed in making the polymerase, and it is immediately found by Randy Saiki to support the PCR process.
With patents submitted, work proceeds for reporting PCR to the general scientific community. An abstract for a meeting in Salt Lake City is submitted in April 1985, and the first announcement of PCR is made there by Saiki in October[20]. Two publications are planned - an 'idea' paper from Mullis, and an 'application' paper from the entire development group. Mullis submits his manuscript to the journal Nature, which rejects it for not including results. The other paper, mainly describing the OR analysis assay, is submitted to Science on September 20, 1985 and is accepted in November. After the rejection of Mullis' report in December, details on the PCR process are hastily added to the second paper, which appears on December 20, 1985[16].
In May of 1986 Mullis presents PCR at the Cold Spring Harbor Symposium[21], and publishes a modified version of his original 'idea' manuscript much later[22]. The first non-Cetus report using PCR is submitted on September 5, 1986[23], indicating how quickly other laboratories are implementing the technique. The Cetus development group publishes their detailed sequence analysis of PCR products on September 8, 1986[15], and their use of ASO probes on November 13, 1986[19].
The use of Taq polymerase in PCR is announced by Henry Erlich at a meeting in Berlin on September 20, 1986, is submitted for publication in October of 1987, and is published early the next year'[24]. The patent for PCR with Taq polymerase is filed on June 17, 1987, and is issued on October 23, 1990[25].
Variation
In December 1985 a joint venture between Cetus and Perkin-Elmer is established to develop instruments and reagents for PCR. Complex Thermal Cyclers are constructed to perform the Klenow-based amplifications, but are never marketed. Simpler machines for Taq-based PCR are developed, and on November 19, 1987 a press release announces the commercial availability of the "PCR-1000 Thermal Cycler" and "AmpliTaq DNA Polymerase".
In the Spring of 1985 John Sninsky at Cetus begins to apply PCR to the difficult task of quantitating the amount of HIV circulating in blood. A viable test is announced on April 11, 1986, and is published in May 1987[26] . Donated blood can now be screened for the virus, and the effect of antiviral drugs can be directly monitored.
In 1985 Norm Arnheim, also a member of the development team, concludes his sabbatical at Cetus and gets a real job at USC. He begins to investigate the use of PCR to amplifiy samples containing just a single copy of the target sequence. By 1989 his lab runs mutiplex-PCR on single sperm to directly analyze the products of meiotic recombination[27]. These single-copy amplifications, which had first been run during the characterization of Taq polymerase[24], become vital to the study of ancient DNA, as well as the genetic typing of preimplanted embryos.
In 1986 Edward Blake, a forensics scientist working in the Cetus building, collaborates with Bruce Budowle (of the FBI) and Cetus researchers to apply PCR to the analysis of criminal evidence. A panel of DNA samples from old cases is collected and coded, and is analyzed blind by Saiki using the HLA DQα assay. When the code is broken, all of the evidence and perpetrators match. Blake uses the technique almost immediately in "Pennsylvania v. Pestinikas"[28], the first use of PCR in a criminal case. This DQα test is developed by Cetus as one of their "Ampli-Type" kits, and goes on to become part of early protocols for the testing of forensic evidence.
By 1989 Alec Jeffreys, who had earlier developed and applied the first DNA Fingerprinting tests, uses PCR to increase their sensitivity[29]. With further modification, the amplification of highly polymorphic VNTR loci will become the standard protocol for National DNA Databases such as CODIS. The guilty go to jail, and the ability of PCR to restest old evidence begins to set the innocent free.
In 1987 Russ Higuchi succeeds in amplifying DNA from a human hair[30]. This work expands to develop methods to amplify DNA from highly degraded samples, such as from Ancient DNA and in forensic evidence. On January 30, 1989 an episode of Star Trek: The Next Generation airs. The ship's doctor is being rapidly aged by a virus attacking her DNA, and is cured when her pre-infection DNA is isolated from a hair found in her cabin. PCR has entered the mainstream media.
Coda
On December 22, 1989 the journal Science awards Taq Polymerase (and PCR) its first "Molecule of the Year". The 'Taq PCR' paper[24] goes on to become (for several years) the most cited publication in biology.
After the publication of the first PCR paper[16], the United States Government sends a stern letter to Randy Saiki, admonishing him for publishing a report on "chain reactions" without the required prior review and approval by the U.S. Department of Energy. Cetus writes back, explaining the differences between PCR and the atomic bomb.
On July 23, 1991 Cetus announces that it will be sold to its neighboring biotechnology company Chiron. As part of the sale, rights to the PCR patents are sold for USD $300 million to Hoffman-La Roche (who in 1989 had bought limited rights to PCR). Many of the Cetus PCR researchers move to a new subsidiary, Roche Molecular Systems.
On October 13, 1993 Kary Mullis, who had left Cetus in 1986, is awarded the Nobel Prize in Chemistry. On the morning of his acceptance speech[1], he is nearly arrested by Swedish authorities for the "inappropriate use of a laser pointer"[31].
References
^ a b c d Kary Mullis' Nobel Lecture, December 8, 1993
^ a b c d e Rabinow P "Making PCR: A Story of Biotechnology" University of Chicago Press (1996) ISBN 0-226-70147-6
^ Watson JD, Crick FHC "A Structure for Deoxyribose Nucleic Acid", Nature vol. 171, pp. 737-738 (1953). [1]
^ (Arthur Kornberg's Discovery of DNA Polymerase I) J. Biol. Chem. vol. 280, p. 46. [2]
^ Lehman, IR, Bessman MJ, Simms ES, Kornberg A "Enzymatic Synthesis of Deoxyribonucleic Acid. I. Preparation of Substrates and Partial Purification of an Enzyme from Escherichia coli" J. Biol. Chem. vol. 233(1) pp. 163-170 (1958).
^ Khorana HG et al. "Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 1. General introduction" J. Biol. Chem. vol. 251(3) pp. 565-70 (1976).
^ Brock TD, Freeze H "Thermus aquaticus, a Nonsporulating Extreme Thermophile" J. Bact. vol. 98(1) pp. 289-297 (1969).
^ Klenow H and Henningsen I "Selective Elimination of the Exonuclease Activity of the Deoxyribonucleic Acid Polymerase from Escherichia coli B by Limited Proteolysis" Proc Natl Acad Sci vol. 65 pp. 168-75 (1970).
^ a b Panet A, Khorana HG "Studies on Polynucleotides" J. Biol. Chem. vol. 249(16), pp. 5213-21 (1974).
^ a b Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG "Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases." J. Molec. Biol. vol. 56, pp. 341-61 (1971).
^ Mullis KB, Ferré F, Gibbs RA "The Polymerase Chain Reaction" Birkhäuser Press (1994) ISBN 0-817-63750-8
^ a b Chien A, Edgar DB, Trela JM "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus" J. Bact. vol. 174 pp. 1550-1557 (1976).
^ Sanger F, Nicklen S, Coulson AR "DNA sequencing with chain-terminating inhibitors" Proc Natl Acad Sci vol. 74(12) pp. 5463-7 (1977).
^ a b c Mullis KB "The Unusual Origins of the Polymerase Chain Reaction" Scientific American, vol. 262, pp. 56-65 (April 1990).
^ a b c d e Scharf et al. "Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences" Science vol. 233, pp. 1076-78 (1986).
^ a b c Saiki RK et al. "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia" Science vol. 230 pp. 1350-54 (1985).
^ Mullis KB "Process for amplifying nucleic acid sequences." U.S. Patent 4,683,202.
^ Mullis, KB et al. "Process for amplifying, detecting, and/or-cloning nucleic acid sequences." U.S. Patent 4,683,195.
^ a b Saiki et al. "Analysis of enzymatically amplified β-globin and HLA DQα DNA with allele-specific oligonucleotide probes." Nature vol. 324 (6093) pp. 163-6 (1986).
^ Saiki, R et al. "A Novel Method for the Prenatal Diagnosis of Sickle Cell Anemia" Amer. Soc. Human Genetics, Oct. 9-13, 1985.
^ Mullis KB et al. "Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction." Cold Spring Harbor Symp. Quant. Biol. vol. 51 pp. 263-73 (1986).
^ Mullis KB and Faloona FA "Specific Synthesis of DNA in vitro via a Polymerase-Catalyzed Chain Reaction." Methods in Enzymology vol. 155(F) pp. 335-50 (1987).
^ Verlaan-de Vries M et al. "A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides." Gene vol. 50(1-3) pp. 313-20 (1986).
^ a b c Saiki et al. "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science vol. 239 pp. 487-91 (1988).
^ Mullis, KB et al. "Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme." U.S. Patent 4,965,188.
^ Kwok S et al. "Identification of HIV sequences by using in vitro enzymatic amplification and oligomer cleavage detection." J. Virol. vol. 61(5) pp. 1690-4 (1987).
^ Boehnke M et al. "Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm." Am J Hum Genet vol. 45(1) pp. 21-32 (1989).
^ Forensic Science Timeline (PDF).
^ Jeffreys A et al. "Amplification of human minisatellites." Nucleic Acids Research vol. 23 pp. 10953-71 (1988).
^ Higuchi R et al. "DNA typing from single hairs." Nature vol. 332(6164) pp. 543-6 (1988).
^ Mullis KB "Dancing Naked in the Mind Field" Pantheon Books (1998) ISBN 0-679-44255-3
Saturday, September 13, 2008
Regular PCR Procedure
General PCR Protocols and Its Product Processes
Recommended Reagent Concentrations
Recommended Reaction Conditions
Initial Conditions
Temperature Cycling
"Hot Start" PCR
Asymmetric PCR for ssDNA Production
Detecting Products
Labelling PCR Products with Digoxigenin
Cleaning PCR Products
Sequencing PCR Products
Cloning PCR Products
AND ALWAYS REMEMBER:
Protocol for PCR using Taq DNA Polymerase
Protocol for PCR with Taq DNA Polymerase. Avoiding Contamination. PCR allows the production of more than 10 million copies of a target DNA sequence from ...www.fermentas.com/techinfo/pcr/dnaamplprotocol.htm -
General PCR Protocol
Detailed PCR protocol from the web site of the Department of Biology, University of Michigan, USA.www.mcdb.lsa.umich.edu/labs/maddock/protocols/PCR/general_pcr_protocol.html
Standard PCR
However, efficient sequencing of dsDNA generated by normal PCR is possible using the modification to the SequenaseTM protocol published by Bachmann et al. ...www.mcb.uct.ac.za/pcrcond.htm
PCR PROTOCOL
PCR PROTOCOL FOR cDNA ARRAYS ON MEMBRANES. Purpose: to amplify insert DNA from purified plasmid DNA derived from bacterial. plasmid libraries. ...www.daf.jhmi.edu/microarray/protocols/protocol6.pdf
Basic PCR Protocol
Basic PCR Protocol. CGLab, 7/2002. 1). Wipe down the bench area with bleach and a new paper towel. 2). Take the PCR components out of the freezer to thaw ...www.sfsu.edu/~biology/cgl/media/PCR%20Protocol-Basic.pdf
Long PCR Protocol
Protocol and guidelines for choice of conditions for PCR of long sequences (10 kb or larger). From Genetics Dept., Harvard Medical School,Boston, MA, USA.arep.med.harvard.edu/labgc/estep/longPCR_protocol.html
20-mer Polymerase Chain Reaction Procedure (for MJ Research ...
MJ Research thermal Cycler: 10-mer PCR for amplification of random genomic DNA fragments ... Edit (or choose a program if it has been set up) PCR Program. ...wheat.pw.usda.gov/~lazo/methods/lazo/pcrproto.html
Single tube confirmation PCR protocol
For characterization colonies of transformed clones of Saccharaomyces, from the web site of the Stanford Genome Technology Center, Palo Alto, CA, USA.www-sequence.stanford.edu/group/yeast_deletion_project/single_tube_protocol.html
Protocol for PCR with Hot Start Taq DNA Polymerase
Protocol for PCR with Hot Start Taq DNA Polymerase. How to Avoid Contamination. During PCR, usually more than 10 million copies of a template DNA can be ...www.fermentas.com/profiles/modifyingenzymes/pdf/protocols/protocolhotstart.pdf
A Basic Polymerase Chain Reaction Protocol
Here, a basic, straight-forward PCR protocol is. presented. Where appropriate, some of the choices for modifying this standard reaction ...www.idtdna.com/support/technical/TechnicalBulletinPDF/A_Basic_PCR_Protocol.pdf
PCR Reamplification Protocol
PCR Reamplification for Inadequate or Failed Amplifications. Change your standard PCR protocol for the locus as follows:. decrease the number of cycles by ...genome-lab.ucdavis.edu/Protocols/pcr_tips/pcr_reamplification.htm
Inverse PCR and Sequencing Protocol
Inverse PCR and Sequencing Protocol on 5 Fly Preps. For recovery of sequences flanking XP elements. This protocol is an adaptation of ...flystocks.bio.indiana.edu/pdfs/Exel_links/5__fly_iPCR_XP_pub.pdf
Recommended Reagent Concentrations
Recommended Reaction Conditions
Initial Conditions
Temperature Cycling
"Hot Start" PCR
Asymmetric PCR for ssDNA Production
Detecting Products
Labelling PCR Products with Digoxigenin
Cleaning PCR Products
Sequencing PCR Products
Cloning PCR Products
AND ALWAYS REMEMBER:
Protocol for PCR using Taq DNA Polymerase
Protocol for PCR with Taq DNA Polymerase. Avoiding Contamination. PCR allows the production of more than 10 million copies of a target DNA sequence from ...www.fermentas.com/techinfo/pcr/dnaamplprotocol.htm -
General PCR Protocol
Detailed PCR protocol from the web site of the Department of Biology, University of Michigan, USA.www.mcdb.lsa.umich.edu/labs/maddock/protocols/PCR/general_pcr_protocol.html
Standard PCR
However, efficient sequencing of dsDNA generated by normal PCR is possible using the modification to the SequenaseTM protocol published by Bachmann et al. ...www.mcb.uct.ac.za/pcrcond.htm
PCR PROTOCOL
PCR PROTOCOL FOR cDNA ARRAYS ON MEMBRANES. Purpose: to amplify insert DNA from purified plasmid DNA derived from bacterial. plasmid libraries. ...www.daf.jhmi.edu/microarray/protocols/protocol6.pdf
Basic PCR Protocol
Basic PCR Protocol. CGLab, 7/2002. 1). Wipe down the bench area with bleach and a new paper towel. 2). Take the PCR components out of the freezer to thaw ...www.sfsu.edu/~biology/cgl/media/PCR%20Protocol-Basic.pdf
Long PCR Protocol
Protocol and guidelines for choice of conditions for PCR of long sequences (10 kb or larger). From Genetics Dept., Harvard Medical School,Boston, MA, USA.arep.med.harvard.edu/labgc/estep/longPCR_protocol.html
20-mer Polymerase Chain Reaction Procedure (for MJ Research ...
MJ Research thermal Cycler: 10-mer PCR for amplification of random genomic DNA fragments ... Edit (or choose a program if it has been set up) PCR Program. ...wheat.pw.usda.gov/~lazo/methods/lazo/pcrproto.html
Single tube confirmation PCR protocol
For characterization colonies of transformed clones of Saccharaomyces, from the web site of the Stanford Genome Technology Center, Palo Alto, CA, USA.www-sequence.stanford.edu/group/yeast_deletion_project/single_tube_protocol.html
Protocol for PCR with Hot Start Taq DNA Polymerase
Protocol for PCR with Hot Start Taq DNA Polymerase. How to Avoid Contamination. During PCR, usually more than 10 million copies of a template DNA can be ...www.fermentas.com/profiles/modifyingenzymes/pdf/protocols/protocolhotstart.pdf
A Basic Polymerase Chain Reaction Protocol
Here, a basic, straight-forward PCR protocol is. presented. Where appropriate, some of the choices for modifying this standard reaction ...www.idtdna.com/support/technical/TechnicalBulletinPDF/A_Basic_PCR_Protocol.pdf
PCR Reamplification Protocol
PCR Reamplification for Inadequate or Failed Amplifications. Change your standard PCR protocol for the locus as follows:. decrease the number of cycles by ...genome-lab.ucdavis.edu/Protocols/pcr_tips/pcr_reamplification.htm
Inverse PCR and Sequencing Protocol
Inverse PCR and Sequencing Protocol on 5 Fly Preps. For recovery of sequences flanking XP elements. This protocol is an adaptation of ...flystocks.bio.indiana.edu/pdfs/Exel_links/5__fly_iPCR_XP_pub.pdf
Videos and Animations for PCR
Genotyping by PCR
Methods for Mouse Genotyping by PCR (protocol 1)
1. Preparation of genomic DNA from the mouse tail.
1) Obtain about 5 mm of the mouse tail and cut it symmetrically into two pieces.
Note: Too long tail can result in the inhibition of PCR because of increased impurity.
Put the cut tail into 500 ul lysis buffer 9see below) in a 1.5 ml microfuge tube, which should be
with a rubber ring to prevent leakage of the content. Without DNA degration, tails can be stored at
-80 centigrade even after standing at room temperature for a couple of hours.
2) Incubate at 65 degree centigrade with gentle shaking overnight. When a part of tail tissue remains
because of inactivation of Proteinase K by the high temperature, addition of more Proteinase K is
recommended to lyse the tail completely.
3)--This step is optional--
Detect the quality of the genomic DNA by 1.0% agarose gel electrophoresis. 10 ul of the lysate is
enough for the detection. The sample may not be suitable for the following PCR unless >4kb DNA
is detected.
4) Heat the lysates at 95 degree centigrade for 10 minutes in a PCR machine or by boiling to inactivate
Proteinase K completely.
5) Spin the tail lysate briefly before transferring to a PCR tube to exclude the tissue debris. Proceed
directly to PCR using the tail DNA lysate as a template at a volume rate of 1/10 as follows.
2. PCR reactions.
Contents of PCR mixture for wildtype/knockout allele screening:
5 ul tail DNA solution: spin briefly before transferring to a PCR tube to avoid contamination of debris.
1 ul 10 uM primers (each upper and lower primer)
5 ul 10x KOD dash DNA polymerase (from TOYOBO Co. LTD.,Japan)
5 ul 2.0 mM dNTPs
32 ul dd H2O
Total volume of 50 ul
We recently found that the final volume can be reduced to 25 ul without mineral oil application.
Sequences of PCR primers: should be designed according to your target gene.
Primers for detecting wild-type allele
Primers for detecting knock-out allele
Methods for Mouse Genotyping by PCR (protocol 2)
Transgenic Genotyping from Tail Biopsies
Harvard University--MCB Department / HSCI
Remove .5-1 cm of the tail and place in 1.5 ml Eppendorf tube. (Store at -20oC until ready to digest).
Digest in Lysis Buffer* + Proteinase K (to 200 ug/ml final conc.).
Incubate in 55oC water bath overnight. (Vortex 1x after 1-2 h).
Add .5 ml Phenol:Chloroform:Isoamyl alcohol (25:24:1) to each tube and vortex for 30 sec.
Spin at top speed in a microcentrifuge for 5 minutes.
Transfer upper (aqueous) phase to new tube; make sure no debris from the interface is transferred.
Add 1 ml of 100% EtOH.
Vortex briefly or shake. Stringy white precipitate (the genomic DNA) should now be visible.
Spin briefly (<1 min) just enough to get the DNA to cling to the plastic, and decant supernatant.
Wash with 1 ml of 70% EtOH.
Let air dry until the pellet becomes partially translucent, but do NOT over-dry, or the DNA will not go into solution any longer.
Redissolve the pellet in 100 ml TE, pH 8.0.
Check concentration, and calculate the total yield, which should be around 10 to 50 mg.
Use 100 ng for subsequent PCR analysis.
*Lysis Buffer:
10 mM Tris-HCl, pH 8.0
25 mM EDTA, pH 8.0
100 mM NaCl
0.5% SDS
DNA from Tail Biopsies
Genotyping Transgenic Rodents by PCR
Isolation of DNA from Mouse Tail Biopsies
Lac-Z Detection in Tail Biopsies
Preparation of Mouse Tail DNA for Dot Blots or PCR
Universal Mouse Genotyping Protocol Using PCR
beta globin Primers
lacZ Primers
neo Primers
1. Preparation of genomic DNA from the mouse tail.
1) Obtain about 5 mm of the mouse tail and cut it symmetrically into two pieces.
Note: Too long tail can result in the inhibition of PCR because of increased impurity.
Put the cut tail into 500 ul lysis buffer 9see below) in a 1.5 ml microfuge tube, which should be
with a rubber ring to prevent leakage of the content. Without DNA degration, tails can be stored at
-80 centigrade even after standing at room temperature for a couple of hours.
2) Incubate at 65 degree centigrade with gentle shaking overnight. When a part of tail tissue remains
because of inactivation of Proteinase K by the high temperature, addition of more Proteinase K is
recommended to lyse the tail completely.
3)--This step is optional--
Detect the quality of the genomic DNA by 1.0% agarose gel electrophoresis. 10 ul of the lysate is
enough for the detection. The sample may not be suitable for the following PCR unless >4kb DNA
is detected.
4) Heat the lysates at 95 degree centigrade for 10 minutes in a PCR machine or by boiling to inactivate
Proteinase K completely.
5) Spin the tail lysate briefly before transferring to a PCR tube to exclude the tissue debris. Proceed
directly to PCR using the tail DNA lysate as a template at a volume rate of 1/10 as follows.
2. PCR reactions.
Contents of PCR mixture for wildtype/knockout allele screening:
5 ul tail DNA solution: spin briefly before transferring to a PCR tube to avoid contamination of debris.
1 ul 10 uM primers (each upper and lower primer)
5 ul 10x KOD dash DNA polymerase (from TOYOBO Co. LTD.,Japan)
5 ul 2.0 mM dNTPs
32 ul dd H2O
Total volume of 50 ul
We recently found that the final volume can be reduced to 25 ul without mineral oil application.
Sequences of PCR primers: should be designed according to your target gene.
Primers for detecting wild-type allele
Primers for detecting knock-out allele
Methods for Mouse Genotyping by PCR (protocol 2)
Transgenic Genotyping from Tail Biopsies
Harvard University--MCB Department / HSCI
Remove .5-1 cm of the tail and place in 1.5 ml Eppendorf tube. (Store at -20oC until ready to digest).
Digest in Lysis Buffer* + Proteinase K (to 200 ug/ml final conc.).
Incubate in 55oC water bath overnight. (Vortex 1x after 1-2 h).
Add .5 ml Phenol:Chloroform:Isoamyl alcohol (25:24:1) to each tube and vortex for 30 sec.
Spin at top speed in a microcentrifuge for 5 minutes.
Transfer upper (aqueous) phase to new tube; make sure no debris from the interface is transferred.
Add 1 ml of 100% EtOH.
Vortex briefly or shake. Stringy white precipitate (the genomic DNA) should now be visible.
Spin briefly (<1 min) just enough to get the DNA to cling to the plastic, and decant supernatant.
Wash with 1 ml of 70% EtOH.
Let air dry until the pellet becomes partially translucent, but do NOT over-dry, or the DNA will not go into solution any longer.
Redissolve the pellet in 100 ml TE, pH 8.0.
Check concentration, and calculate the total yield, which should be around 10 to 50 mg.
Use 100 ng for subsequent PCR analysis.
*Lysis Buffer:
10 mM Tris-HCl, pH 8.0
25 mM EDTA, pH 8.0
100 mM NaCl
0.5% SDS
DNA from Tail Biopsies
Genotyping Transgenic Rodents by PCR
Isolation of DNA from Mouse Tail Biopsies
Lac-Z Detection in Tail Biopsies
Preparation of Mouse Tail DNA for Dot Blots or PCR
Universal Mouse Genotyping Protocol Using PCR
beta globin Primers
lacZ Primers
neo Primers
PCR Primer Design Tools
PCR Troubleshooting
Ten Things That Can Kill Your PCR
Ten Things That Can Kill Your PCR. by Peter Frame. A blank PCR gel has got to be one of the most aggravating things about. molecular biology. ...www.mbi.ufl.edu/~rowland/protocols/pcr.htm
PCR trouble shooting, help, suggestions and advice
PCR trouble shooting, help, suggestions and advice. If your PCR amplification somehow performs unexpectedly, it is usually caused by one of the listed ...biologi.uio.no/bot/ascomycetes/PCR.troubleshooting.html
PCR Troubleshooting
Troubleshooting PCR. Polymerase Chain Reaction problems and solutions, PCR help.www.pcrstation.com/pcr-troubleshooting/
Troubleshooting Guide
MultiplexPCR Troubleshooting Guide. Poor amplification of some or all loci. Pipetting error /. reagents missing. Repeat experiment checking the ...
www.abgene.com/downloads/Guide_PCR-multiplex-v2-0208.pdf
PCR-Online.org - PCR Protocols, Troubleshooting and Information
Westernblotting.org: definitions, molecular biology links, protocols, troubleshooting and technical information for those interested in western blots and...
www.pcr-online.org/Troubleshooting.htm
PCR troubleshooting - MyBio
PCR troubleshooting - Web Resources. Optimizing DNA Amplification Protocols Optimizing DNA Amplification Protocols using the Eppendorf ?? Mastercycler ?? ...mybio.net/biowiki/PCR_troubleshooting
Troubleshooting PCR Why do I have non-specific bands when I run my ...
Appendix III:Troubleshooting. Successful PCR Guide. Takara Mirus Bio. 38. Causes.Trouble-shooting measures. Concentration of primers is too high ...www.takarabiousa.com/docs/PCR_TRBSHT.pdf -
Troubleshooting the PCR procedure Specific application of PCR ...
Troubleshooting the. PCR procedure. For a detailed discussion of the factors that. influence PCR and how to troubleshoot the ...www.roche-applied-science.com/PROD_INF/MANUALS/epitope/p18-19.pdf
Optimization and troubleshooting in PCR.
Optimization and troubleshooting in PCR. References. http://www.genome.org#References. This article cites 42 articles, 13 of which can be accessed free at: ...www.genome.org/cgi/reprint/4/5/S185.pdf -
EdgeBio ExcelaPure 96-Well UF PCR Purification Kit Troubleshooting ...
Troubleshooting Guide forExcelaPure 96-Well UF PCR Purification Kit>www.edgebio.com/tech/tsg/ExcelaPure96-wellUF_TSG.html
Ten Things That Can Kill Your PCR. by Peter Frame. A blank PCR gel has got to be one of the most aggravating things about. molecular biology. ...www.mbi.ufl.edu/~rowland/protocols/pcr.htm
PCR trouble shooting, help, suggestions and advice
PCR trouble shooting, help, suggestions and advice. If your PCR amplification somehow performs unexpectedly, it is usually caused by one of the listed ...biologi.uio.no/bot/ascomycetes/PCR.troubleshooting.html
PCR Troubleshooting
Troubleshooting PCR. Polymerase Chain Reaction problems and solutions, PCR help.www.pcrstation.com/pcr-troubleshooting/
Troubleshooting Guide
MultiplexPCR Troubleshooting Guide. Poor amplification of some or all loci. Pipetting error /. reagents missing. Repeat experiment checking the ...
www.abgene.com/downloads/Guide_PCR-multiplex-v2-0208.pdf
PCR-Online.org - PCR Protocols, Troubleshooting and Information
Westernblotting.org: definitions, molecular biology links, protocols, troubleshooting and technical information for those interested in western blots and...
www.pcr-online.org/Troubleshooting.htm
PCR troubleshooting - MyBio
PCR troubleshooting - Web Resources. Optimizing DNA Amplification Protocols Optimizing DNA Amplification Protocols using the Eppendorf ?? Mastercycler ?? ...mybio.net/biowiki/PCR_troubleshooting
Troubleshooting PCR Why do I have non-specific bands when I run my ...
Appendix III:Troubleshooting. Successful PCR Guide. Takara Mirus Bio. 38. Causes.Trouble-shooting measures. Concentration of primers is too high ...www.takarabiousa.com/docs/PCR_TRBSHT.pdf -
Troubleshooting the PCR procedure Specific application of PCR ...
Troubleshooting the. PCR procedure. For a detailed discussion of the factors that. influence PCR and how to troubleshoot the ...www.roche-applied-science.com/PROD_INF/MANUALS/epitope/p18-19.pdf
Optimization and troubleshooting in PCR.
Optimization and troubleshooting in PCR. References. http://www.genome.org#References. This article cites 42 articles, 13 of which can be accessed free at: ...www.genome.org/cgi/reprint/4/5/S185.pdf -
EdgeBio ExcelaPure 96-Well UF PCR Purification Kit Troubleshooting ...
Troubleshooting Guide forExcelaPure 96-Well UF PCR Purification Kit>www.edgebio.com/tech/tsg/ExcelaPure96-wellUF_TSG.html
Wednesday, September 10, 2008
About PCR
1. IntroductionIn 1983 Kary B. Mullis was driving through California on a moonlight night (Mullis, 1990). He was pondering how to use DNA polymerase with oligonucleotide primers in order to identify a given nucleotide at a given position in a complex DNA molecule, such as the human genome. During this drive he invented or discovered the elegant method of making unlimited DNA copies from a single copy of DNA, and called the method: "Polymerase Chain Reaction" (PCR). A couple of months later he conducted the first successful experiment. Ten years after his drive in California, he was awarded the Nobel Prize in Stockholm for his brilliant discovery (Carr, 1993).
PCR was first published in 1985 (Saiki et al., 1985) with Klenow polymerase used as the elongation enzyme. Due to the heat instability of the Klenow polymerase, new enzyme had to be added for every new cycle, and the maximum limit of the product length was 400 bp. In 1988 the first report using DNA polymerase from Thermophilus aquaticus (Taq-polymerase) was published (Saiki et al., 1988). This polymerase greatly enhanced the value of PCR, and the introduction of the automatic programmable heating block in the same report also took the tedious need for three different water baths out of the procedure. Currently the PCR technique is utilized in most molecular biology laboratories as a routine tool which is suitable for performing a great number of different experiments. The method is frequently chosen for conducting experiments, such as cloning, making mutations, sequencing, detecting, typing, etc. (Erlich et al., 1991).
2. AnimationThe basic molecular events of PCR are illustrated in an animation of the liquid phase DNA amplification, which is a prerequisite of the solid phase DNA amplification. The whole animation can be seen in the DIAPOPS animation.
3. The basic reactionPCR is based on the recognition by a short piece of DNA (the primer) of a sequence on a larger, single stranded fragment of DNA (template strand). When the primer recognizes the template and binds (anneals) to the recognition sequence, the 3'-end of the primer is used by DNA polymerase to synthesize a new DNA strand (elongation). When the temperature is raised, the new DNA strand will melt away (denature) from the template, and the template is once again open for annealing of a new primer when the temperature is decreased. By adding a second primer which recognizes the template strand complementary to the first template, the elongation can proceed in the direction of the first primer. In the first round of elongation, this will ideally double the amount of template strands. In the second temperature cycling, half of the templates for the first primer will be new-synthesized fragments, all terminated where the second primer annealed. When these new fragments are recognized by the first primer, the elongation cannot proceed beyond the second primer, and the synthesized fragments will have a fixed length determined by the distance of the annealing sites of the two primers. New production of template strands take place in every temperature cycle. In this way the DNA sequence between the two primer sequences is amplified exponentially, yielding high concentrations of double-stranded DNA of the same length. The newly-formed double stranded DNA is denatured at 94-97ºC. Primers anneal at 35-72ºC (the exact temperature is primer- and assay dependent), and the new product is synthesized at 72ºC, which is the optimal temperature for the Taq-polymerase.
4. ConclusionPCR is capable of producing large amounts of DNA fragments from a single piece of template DNA as the amplification increases the amount of fragments produced exponentially. In theory, it is possible to detect a single copy of template DNA by PCR using simple methods. For this reason PCR is used to identify nucleic acid sequences that are only present in very small numbers in the sample to be analyzed.
Lecture of PCR-2
Introduction to PCR. Molecular biology relies on techniques that enable the detection or ... With the introduction of the Polymerase Chain Reaction (PCR), ...www.modares.ac.ir/elearning/mnaderi/Genetic%20Engineering%20course%20II/Pages/Lecture2.htm
PCR Technology
Introduction. Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in molecular biology and for good reason: it is a ...www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.html
Introduction to PCR
Either way, the DNA is extracted from the source and is amplified via PCR (the Polymerase Chain Reaction). This allows very minute amounts of DNA to be ...nature.umesci.maine.edu/forensics/p_intro.htm
6.1 Polymerase Chain Reaction (PCR) Introduction6.1 Polymerase Chain Reaction (PCR). Introduction. T. he polymerase chain reaction technique employs oligonucleotide primers to amplify segments of ...www.fws.gov/policy/library/fh_handbook/Volume_1/Chapter_6.pdf
Real-Time PCR Introduction [M.Tevfik DORAK]
Overview by MT Dorak, University of Alabama at Birmingham, USA.dorakmt.tripod.com/genetics/realtime.html
YouTube - EDIROL PCR Introduction
This is a video introduction to our new PCR MIDI controllers.www.youtube.com/watch?v=vfiK7Fl75ZQ
PCR was first published in 1985 (Saiki et al., 1985) with Klenow polymerase used as the elongation enzyme. Due to the heat instability of the Klenow polymerase, new enzyme had to be added for every new cycle, and the maximum limit of the product length was 400 bp. In 1988 the first report using DNA polymerase from Thermophilus aquaticus (Taq-polymerase) was published (Saiki et al., 1988). This polymerase greatly enhanced the value of PCR, and the introduction of the automatic programmable heating block in the same report also took the tedious need for three different water baths out of the procedure. Currently the PCR technique is utilized in most molecular biology laboratories as a routine tool which is suitable for performing a great number of different experiments. The method is frequently chosen for conducting experiments, such as cloning, making mutations, sequencing, detecting, typing, etc. (Erlich et al., 1991).
2. AnimationThe basic molecular events of PCR are illustrated in an animation of the liquid phase DNA amplification, which is a prerequisite of the solid phase DNA amplification. The whole animation can be seen in the DIAPOPS animation.
3. The basic reactionPCR is based on the recognition by a short piece of DNA (the primer) of a sequence on a larger, single stranded fragment of DNA (template strand). When the primer recognizes the template and binds (anneals) to the recognition sequence, the 3'-end of the primer is used by DNA polymerase to synthesize a new DNA strand (elongation). When the temperature is raised, the new DNA strand will melt away (denature) from the template, and the template is once again open for annealing of a new primer when the temperature is decreased. By adding a second primer which recognizes the template strand complementary to the first template, the elongation can proceed in the direction of the first primer. In the first round of elongation, this will ideally double the amount of template strands. In the second temperature cycling, half of the templates for the first primer will be new-synthesized fragments, all terminated where the second primer annealed. When these new fragments are recognized by the first primer, the elongation cannot proceed beyond the second primer, and the synthesized fragments will have a fixed length determined by the distance of the annealing sites of the two primers. New production of template strands take place in every temperature cycle. In this way the DNA sequence between the two primer sequences is amplified exponentially, yielding high concentrations of double-stranded DNA of the same length. The newly-formed double stranded DNA is denatured at 94-97ºC. Primers anneal at 35-72ºC (the exact temperature is primer- and assay dependent), and the new product is synthesized at 72ºC, which is the optimal temperature for the Taq-polymerase.
4. ConclusionPCR is capable of producing large amounts of DNA fragments from a single piece of template DNA as the amplification increases the amount of fragments produced exponentially. In theory, it is possible to detect a single copy of template DNA by PCR using simple methods. For this reason PCR is used to identify nucleic acid sequences that are only present in very small numbers in the sample to be analyzed.
Lecture of PCR-2
Introduction to PCR. Molecular biology relies on techniques that enable the detection or ... With the introduction of the Polymerase Chain Reaction (PCR), ...www.modares.ac.ir/elearning/mnaderi/Genetic%20Engineering%20course%20II/Pages/Lecture2.htm
PCR Technology
Introduction. Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in molecular biology and for good reason: it is a ...www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.html
Introduction to PCR
Either way, the DNA is extracted from the source and is amplified via PCR (the Polymerase Chain Reaction). This allows very minute amounts of DNA to be ...nature.umesci.maine.edu/forensics/p_intro.htm
6.1 Polymerase Chain Reaction (PCR) Introduction6.1 Polymerase Chain Reaction (PCR). Introduction. T. he polymerase chain reaction technique employs oligonucleotide primers to amplify segments of ...www.fws.gov/policy/library/fh_handbook/Volume_1/Chapter_6.pdf
Real-Time PCR Introduction [M.Tevfik DORAK]
Overview by MT Dorak, University of Alabama at Birmingham, USA.dorakmt.tripod.com/genetics/realtime.html
YouTube - EDIROL PCR Introduction
This is a video introduction to our new PCR MIDI controllers.www.youtube.com/watch?v=vfiK7Fl75ZQ
Introduction to PCR
PCR—from (Dr. Chen, Dept of Biochem. & Mol. Biology, Univ. College London)
Polymerase Chain Reaction
1) Add the following to a microfuge tube:10 ul reaction buffer1 ul 15 uM forward primer1 ul 15 uM reverse primer1 ul template DNA5 ul 2 mM dNTP8 ul 25 mM MgCl2 or MgSO4 (volume variable)water (to make up to 100 ul)
2) Place tube in a thermocycler. Heat sample to 95C, then add 0.5 -1 ul of enzyme (Taq, Tli, Pfu etc.). Add a few drops of mineral oil.
3) Start the PCR cycles according the following schemes:
a) denaturation - 94C, 30-90 sec.b) annealing - 55C (or -5C Tm), 0.5-2 min. c) extension - 72C, 1 min. (time depends on length of PCR product and enzyme used)repeat cycles 29 times
4) Add a final extension step of 5 min. to fill in any uncompleted polymerisation. Then cooled down to 4- 25C.
Note: Most of the parameters can be varied to optimise the PCR (more at Tavi's PCR guide):a) Mg++ - one of the main variables - change the amount added if the PCR result is poor. Mg++ affects the annealing of the oligo to the template DNA by stabilising the oligo-template interaction, it also stabilises the replication complex of polymerase with template-primer. It can therefore also increases non-specific annealing and produced undesirable PCR products (gives multiple bands in gel). EDTA which chelate Mg++ can change the Mg++ concentration.b) Template DNA concentration - PCR is very powerful tool for DNA amplification therefore very little DNA is needed. But to reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used, though too much template may increase the amount of contaminants and reduce efficiency.c) Enzymes used - Taq DNA polymerase has a higher error rate (no proof-reading 3' to 5' exonuclease activity) than Tli or Pfu. Use Tli, Pfu or other polymerases with good proof-reading capability if high fidelity is needed. Taq, however, is less fussy than other polymerases and less likely to fail. It can be used in combination with other enzymes to increase its fidelity. Taq also tends to add extra A's at the 3'end (extra A's are useful for TA cloning but needs to be removed if blunt end ligation is to be done). More enzymes can also be added to improve efficiency (since Taq may be damaged in repeated cycling) but may increase non-specific PCR products. Vent polymerase may degrade primer and therefore not ideal for mutagenesis-by-PCR work. d) dNTP - can use up to 1.5 mM dNTP. dNTP chelate Mg++, therefore amount of Mg++ used may need to be changed. However excessive dNTP can increase the error rate and possibly inhibits Taq. Lowering the dNTP (10-50 uM) may therefore also reduce error rate. Larger size PCR fragment need more dNTP. e) primers - up to 3 uM of primers may be used, but high primer to template ratio can results in non-specific amplification and primer-dimer formation (note: store primers in small aliquots). f) Primer design - check primer sequences to avoid primer-dimer formation. Add a GC-clamp at the 5' end if a restriction site is introduced there. One or two G or C at the 3' end is fine but try to avoid having too many (it can result in non-specific PCR products). Perfect complementarity of 18 bases or more is ideal. See Guide.g) Thermal cycling - denaturation time can be increased if template GC content is high. Higher annealing temperature may be needed for primers with high GC content or longer primers (calculate Tm). Using a gradient (if your PCR machine permits it) is a useful way of determining the annealing temperature. Extension time should be extended for larger PCR products; but reduced it whenever possible to limit damage to enzyme. Extension time is also affected by the enzymes used e.g for Taq - assume 1000 base/min (also check suppliers' recommendations, actual rate is much higher). The number of cycle can be increased if the number of template DNA is very low, and decreased if high amount of template DNA is used (higher template DNA is preferable for PCR cloning - lower error rate in the PCR).
h) Additives -
Glycerol (5-10%), formamide (1-5%) or DMSO (2-10%) can be added in PCR for template DNA with high GC content (they change the Tm of primer-template hybridisation reaction and the thermostability of polymerase enzyme). Glycerol can protects Taq against heat damage, while formamide may lower enzyme resistence.
0.5 -2M Betaine (stock solution - 5M) is also useful for PCR over high GC content and long stretches of DNA (Long PCR / LA PCR). Perform a titration to determine to optimum concentration (1.3 M recommended). Reduce melting temperature (92 -93 °C) and annealing temperature (1-2°C lower). It may be useful to use betaine in combination with other reagents like 5%DMSO. Betaine is often the secret (and unnecessarily expensive) ingredient of many commercial kits.
>50mM TMAC (tetramethylammonium chloride), TEAC (tetraethylammonium chloride), and TMANO (trimethlamine N-oxide) can also be used.
BSA (up to 0.8 µg/µl) can also improve efficiency of PCR reaction.
See also Dan Cruickshank's PCR additives and Alkami Enhancers for more.
i) PCR buffer
Higher concentration of PCR buffer may be used to improve efficiency.
This buffer may work better than the buffer supplied from commercial sources.16.6 mM ammonium sulfate67.7 mM TRIS-HCl, pH 8.8910 mM beta-mercaptoethanol170 micrograms/ml BSA1.5-3 mM MgCl2
j) The PCR product may be purified using a number of commercially available products or by gel-purification if the template needed to be removed. It can also be sequenced.
k) Trouble shooting see Tavi's page, MycoSite, Alkami Biosystems, Promega and Sigma.
l) PCR methods
Hot-start PCR - to reduce non-specific amplification. Can also be done by separating the DNA mixtures from enzyme by a layer of wax which melts when heated in cycling reaction. A number of companies also produce hot start PCR products, See Alkami Biosystem.
"Touch-down" PCR - start at high annealing temperature, then decrease annealing temperature in steps to reduce non-specific PCR product. Can also be used to determine DNA sequence of known protein sequence.
Nested PCR - use to synthesize more reliable product - PCR using a outer set of primers and the product of this PCR is used for further PCR reaction using an inner set of primers.
Inverse PCR - for amplification of regions flanking a known sequence. DNA is digested, the desired fragment is circularise by ligation, then PCR using primer complementary to the known sequence extending outwards.
AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA) - methods for creating genomic fingerprints from species with little-known target sequences by amplifying using arbitrary oligonucleotides. It is normally done at low and then high stringency to determine the relatedness of species or for analysis of Restriction Fragment Length Polymorphisms (RFLP).
RT-PCR (reverse transcriptase) - using RNA-directed DNA polymerase to synthesize cDNAs which is then used for PCR and is extremely sensitive for detecting the expression of a specific sequence in a tissue or cells. It may also be use to quantify mRNA transcripts. See also Quantiative RT-PCR, Competitive Quantitative RT-PCR, RT in situ PCR, Nested RT-PCR.
RACE (rapid amplificaton of cDNA ends) - used where information about DNA/protein sequence is limited. Amplify 3' or 5' ends of cDNAs generating fragments of cDNA with only one specific primer each (+ one adaptor primer). Overlapping RACE products can then be combined to produce full cDNA. See also Gibco manual.
DD-PCR (differential display) - used to identify differentially expressed genes in different tissues. First step involves RT-PCR, then amplification using short, intentionally nonspecific primers. Get series of band in a high-resolution gel and compare to that from other tissues, any bands unique to single samples are considered to be differentially expressed.
Multiplex-PCR - 2 or more unique targets of DNA sequences in the same specimen are amplified simultaneously. One can be use as control to verify the integrity of PCR. Can be used for mutational analysis and identification of pathogens.
Q/C-PCR (Quantitative comparative) - uses an internal control DNA sequence (but of different size) which compete with the target DNA (competitive PCR) for the same set of primers. Used to determint the amount of target template in the reaction.
Recusive PCR - Used to synthesise genes. Oligos used are complementary to stretches of a gene (>80 bases), alternately to the sense and to the antisense strands with ends overlapping (~20 bases). Design of the oligo avoiding homologous sequence (>8) is crucial to the success of this method.
Asymmetric PCR
In Situ PCR
Mutagenesis by PCR
Far too many to list properly.
For more information, protocols and links, go to PCR jump station, Alkami Biosystem, Fermentas, Promega, and Sigma, See also PCR primer, PCR notes and PCR manual at Roche and Qiagen.
Other PCR links - PCR lectures, radio-labelled probes, Thermocycler suppliers
Polymerase Chain Reaction
1) Add the following to a microfuge tube:10 ul reaction buffer1 ul 15 uM forward primer1 ul 15 uM reverse primer1 ul template DNA5 ul 2 mM dNTP8 ul 25 mM MgCl2 or MgSO4 (volume variable)water (to make up to 100 ul)
2) Place tube in a thermocycler. Heat sample to 95C, then add 0.5 -1 ul of enzyme (Taq, Tli, Pfu etc.). Add a few drops of mineral oil.
3) Start the PCR cycles according the following schemes:
a) denaturation - 94C, 30-90 sec.b) annealing - 55C (or -5C Tm), 0.5-2 min. c) extension - 72C, 1 min. (time depends on length of PCR product and enzyme used)repeat cycles 29 times
4) Add a final extension step of 5 min. to fill in any uncompleted polymerisation. Then cooled down to 4- 25C.
Note: Most of the parameters can be varied to optimise the PCR (more at Tavi's PCR guide):a) Mg++ - one of the main variables - change the amount added if the PCR result is poor. Mg++ affects the annealing of the oligo to the template DNA by stabilising the oligo-template interaction, it also stabilises the replication complex of polymerase with template-primer. It can therefore also increases non-specific annealing and produced undesirable PCR products (gives multiple bands in gel). EDTA which chelate Mg++ can change the Mg++ concentration.b) Template DNA concentration - PCR is very powerful tool for DNA amplification therefore very little DNA is needed. But to reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used, though too much template may increase the amount of contaminants and reduce efficiency.c) Enzymes used - Taq DNA polymerase has a higher error rate (no proof-reading 3' to 5' exonuclease activity) than Tli or Pfu. Use Tli, Pfu or other polymerases with good proof-reading capability if high fidelity is needed. Taq, however, is less fussy than other polymerases and less likely to fail. It can be used in combination with other enzymes to increase its fidelity. Taq also tends to add extra A's at the 3'end (extra A's are useful for TA cloning but needs to be removed if blunt end ligation is to be done). More enzymes can also be added to improve efficiency (since Taq may be damaged in repeated cycling) but may increase non-specific PCR products. Vent polymerase may degrade primer and therefore not ideal for mutagenesis-by-PCR work. d) dNTP - can use up to 1.5 mM dNTP. dNTP chelate Mg++, therefore amount of Mg++ used may need to be changed. However excessive dNTP can increase the error rate and possibly inhibits Taq. Lowering the dNTP (10-50 uM) may therefore also reduce error rate. Larger size PCR fragment need more dNTP. e) primers - up to 3 uM of primers may be used, but high primer to template ratio can results in non-specific amplification and primer-dimer formation (note: store primers in small aliquots). f) Primer design - check primer sequences to avoid primer-dimer formation. Add a GC-clamp at the 5' end if a restriction site is introduced there. One or two G or C at the 3' end is fine but try to avoid having too many (it can result in non-specific PCR products). Perfect complementarity of 18 bases or more is ideal. See Guide.g) Thermal cycling - denaturation time can be increased if template GC content is high. Higher annealing temperature may be needed for primers with high GC content or longer primers (calculate Tm). Using a gradient (if your PCR machine permits it) is a useful way of determining the annealing temperature. Extension time should be extended for larger PCR products; but reduced it whenever possible to limit damage to enzyme. Extension time is also affected by the enzymes used e.g for Taq - assume 1000 base/min (also check suppliers' recommendations, actual rate is much higher). The number of cycle can be increased if the number of template DNA is very low, and decreased if high amount of template DNA is used (higher template DNA is preferable for PCR cloning - lower error rate in the PCR).
h) Additives -
Glycerol (5-10%), formamide (1-5%) or DMSO (2-10%) can be added in PCR for template DNA with high GC content (they change the Tm of primer-template hybridisation reaction and the thermostability of polymerase enzyme). Glycerol can protects Taq against heat damage, while formamide may lower enzyme resistence.
0.5 -2M Betaine (stock solution - 5M) is also useful for PCR over high GC content and long stretches of DNA (Long PCR / LA PCR). Perform a titration to determine to optimum concentration (1.3 M recommended). Reduce melting temperature (92 -93 °C) and annealing temperature (1-2°C lower). It may be useful to use betaine in combination with other reagents like 5%DMSO. Betaine is often the secret (and unnecessarily expensive) ingredient of many commercial kits.
>50mM TMAC (tetramethylammonium chloride), TEAC (tetraethylammonium chloride), and TMANO (trimethlamine N-oxide) can also be used.
BSA (up to 0.8 µg/µl) can also improve efficiency of PCR reaction.
See also Dan Cruickshank's PCR additives and Alkami Enhancers for more.
i) PCR buffer
Higher concentration of PCR buffer may be used to improve efficiency.
This buffer may work better than the buffer supplied from commercial sources.16.6 mM ammonium sulfate67.7 mM TRIS-HCl, pH 8.8910 mM beta-mercaptoethanol170 micrograms/ml BSA1.5-3 mM MgCl2
j) The PCR product may be purified using a number of commercially available products or by gel-purification if the template needed to be removed. It can also be sequenced.
k) Trouble shooting see Tavi's page, MycoSite, Alkami Biosystems, Promega and Sigma.
l) PCR methods
Hot-start PCR - to reduce non-specific amplification. Can also be done by separating the DNA mixtures from enzyme by a layer of wax which melts when heated in cycling reaction. A number of companies also produce hot start PCR products, See Alkami Biosystem.
"Touch-down" PCR - start at high annealing temperature, then decrease annealing temperature in steps to reduce non-specific PCR product. Can also be used to determine DNA sequence of known protein sequence.
Nested PCR - use to synthesize more reliable product - PCR using a outer set of primers and the product of this PCR is used for further PCR reaction using an inner set of primers.
Inverse PCR - for amplification of regions flanking a known sequence. DNA is digested, the desired fragment is circularise by ligation, then PCR using primer complementary to the known sequence extending outwards.
AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA) - methods for creating genomic fingerprints from species with little-known target sequences by amplifying using arbitrary oligonucleotides. It is normally done at low and then high stringency to determine the relatedness of species or for analysis of Restriction Fragment Length Polymorphisms (RFLP).
RT-PCR (reverse transcriptase) - using RNA-directed DNA polymerase to synthesize cDNAs which is then used for PCR and is extremely sensitive for detecting the expression of a specific sequence in a tissue or cells. It may also be use to quantify mRNA transcripts. See also Quantiative RT-PCR, Competitive Quantitative RT-PCR, RT in situ PCR, Nested RT-PCR.
RACE (rapid amplificaton of cDNA ends) - used where information about DNA/protein sequence is limited. Amplify 3' or 5' ends of cDNAs generating fragments of cDNA with only one specific primer each (+ one adaptor primer). Overlapping RACE products can then be combined to produce full cDNA. See also Gibco manual.
DD-PCR (differential display) - used to identify differentially expressed genes in different tissues. First step involves RT-PCR, then amplification using short, intentionally nonspecific primers. Get series of band in a high-resolution gel and compare to that from other tissues, any bands unique to single samples are considered to be differentially expressed.
Multiplex-PCR - 2 or more unique targets of DNA sequences in the same specimen are amplified simultaneously. One can be use as control to verify the integrity of PCR. Can be used for mutational analysis and identification of pathogens.
Q/C-PCR (Quantitative comparative) - uses an internal control DNA sequence (but of different size) which compete with the target DNA (competitive PCR) for the same set of primers. Used to determint the amount of target template in the reaction.
Recusive PCR - Used to synthesise genes. Oligos used are complementary to stretches of a gene (>80 bases), alternately to the sense and to the antisense strands with ends overlapping (~20 bases). Design of the oligo avoiding homologous sequence (>8) is crucial to the success of this method.
Asymmetric PCR
In Situ PCR
Mutagenesis by PCR
Far too many to list properly.
For more information, protocols and links, go to PCR jump station, Alkami Biosystem, Fermentas, Promega, and Sigma, See also PCR primer, PCR notes and PCR manual at Roche and Qiagen.
Other PCR links - PCR lectures, radio-labelled probes, Thermocycler suppliers
Polymerase chain reaction--PCR
From Wikipedia, the free encyclopedia
"PCR" redirects here. For other uses, see PCR (disambiguation).
A strip of eight PCR tubes, each containing a 100μl reaction.
The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
Developed in 1983 by Kary Mullis,[1] PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.[2][3] These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis won the Nobel Prize in Chemistry for his work on PCR.[4]
Contents
1 PCR principles and procedure
1.1 Procedure
2 PCR stages
2.1 PCR optimization
3 Application of PCR
3.1 Isolation of genomic DNA
3.2 Amplification and quantitation of DNA
3.3 PCR in diagnosis of diseases
4 Variations on the basic PCR technique
5 History
5.1 Patent wars
6 References
7 External links
PCR principles and procedure
PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.[5]
A basic PCR set up requires several components and reagents.[6] These components include:
DNA template that contains the DNA region (target) to be amplified.
Two primers, which are complementary to the DNA regions at the 5' (five prime) or 3' (three prime) ends of the DNA region.
A DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C.
Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis[7]
Monovalent cation potassium ions.
The PCR is commonly carried out in a reaction volume of 20-150 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.
Procedure
Schematic drawing of the PCR cycle. (1) Denaturing at 94-96°C. (2) Annealing at ~65°C (3) Elongation at 72°C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses.
The PCR usually consists of a series of 20 to 40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps (Fig. 2). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.[8]
Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.[9]
Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C,[10][11] and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.
Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.
To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products.
PCR stages
The PCR process can be divided into three stages:
Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very specific and precise.[citation needed]
Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.
Plateau: No more product accumulates due to exhaustion of reagents and enzyme.
PCR optimization
Main article: PCR optimization
In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.[12][13] Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.[6] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA.
Application of PCR
Isolation of genomic DNA
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.
Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constructs[14]. PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.
Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing. This technique may also be used to determine evolutionary relationships among organisms.
Amplification and quantitation of DNA
Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.[15]
Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample – a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.
See also Use of DNA in forensic entomology
PCR in diagnosis of diseases
PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely.[citation needed] PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.[citation needed]
PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.[citation needed]
Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below).
Variations on the basic PCR technique
Main article: Variants of PCR
Allele-specific PCR: This diagnostic or cloning technique is used to identify or utilize single-nucleotide polymorphisms (SNPs) (single base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.[16] See SNP genotyping for more information.
Assembly PCR or Polymerase Cycling Assembly (PCA): Assembly PCR is the artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final long DNA product.[17]
Asymmetric PCR: Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.[18] A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Melting temperatureTm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.[19]
Helicase-dependent amplification: This technique is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.[20]
Hot-start PCR: This is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase.[21] Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody[9] or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
Intersequence-specific (ISSR) PCR: a PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.[22]
Inverse PCR: a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.[23]
Ligation-mediated PCR: This method uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.[24]
Methylation-specific PCR (MSP): The MSP method was developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine,[25] and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
Miniprimer PCR: Miniprimer PCR uses a novel thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides, instead of the approximately 20 nucleotides required by Taq. This method permits PCR targeting smaller primer binding regions, and is particularly useful to amplify unknown, but conserved, DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene. 16S rRNA miniprimer PCR was used to characterize a microbial mat community growing in an extreme environment, a hypersaline pond in Puerto Rico. In that study, deeply divergent sequences were discovered with high frequency and included representatives that defined two new division-level taxa, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity.[26] By enlarging the "sequence space" that may be queried by PCR primers, this technique may enable novel PCR strategies that are not possible within the limits of primer design imposed by Taq and other commonly used enzymes.
Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).
Multiplex-PCR: The use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
Overlap-extension PCR: is a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.
Quantitative PCR (Q-PCR): is used to measure the quantity of a PCR product (preferably real-time). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR commonly refers to reverse transcription PCR (see below), which is often used in conjunction with Q-PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.
RT-PCR: (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method, named RACE-PCR, short for Rapid Amplification of cDNA Ends.
Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (the only primers present are covalently linked to solid support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR[27] (where conventional Solid Phase PCR can be improved by employing high Tm solid support primer with application of a thermal 'step' to favour solid support priming).
TAIL-PCR: Thermal asymmetric interlaced PCR is used to isolate unknown sequence flanking a known sequence. Within the known sequence TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.[28]
Touchdown PCR: a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5˚C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5˚C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.[29]
PAN-AC: This method uses isothermal conditions for amplification, and may be used in living cells.[30][31]
Universal Fast Walking: this method allows genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer - which can lead to artefactual 'noise') [32] by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends) [33], 5'RACE LaNe [34] and 3'RACE LaNe [35].
History
Main article: History of polymerase chain reaction
A 1971 paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro.[36] However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.[37]
At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90°C (>195°F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures.[2] So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.
A 1976 discovery of Taq polymerase a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F)) environments[10] paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation,[11] thus obviating the need to add new DNA polymerase after each cycle[3]. This allowed an automated thermocycler-based process for DNA amplification.
At the time he developed PCR in 1983, Mullis was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car.[38] He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase.
In Scientific American, Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."[39] He was awarded the Nobel Prize in Chemistry in 1993 for his invention,[4] seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis' work, and whether he had been the sole inventor of the PCR principle. (see main article: Kary Mullis)
Patent wars
The PCR technique was patented by Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.
A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega. The legal arguments have extended beyond the life of the original PCR and Taq polymerase patents, which expired on March 28, 2005[40]
References
^ Bartlett & Stirling (2003)—A Short History of the Polymerase Chain Reaction. In: Methods Mol Biol. 226:3-6
^ a b Saiki, RK; Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (Dec 20 1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science 230 (4732): 1350–4. doi:10.1126/science.2999980. PMID 2999980.
^ a b Saiki, RK; Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science 239: 487–91. doi:10.1126/science.2448875. PMID 2448875.
^ a b Karry Mullis Nobel Lecture, December 8, 1993
^ Cheng S, Fockler C, Barnes WM, Higuchi R (1994). "Effective amplification of long targets from cloned inserts and human genomic DNA". Proc Natl Acad Sci. 91: 5695–5699. doi:10.1073/pnas.91.12.5695. PMID 8202550.
^ a b Joseph Sambrook and David W. Russel (2001). Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. ISBN 0-87969-576-5. Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction
^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004). "Recent developments in the optimization of thermostable DNA polymerases for efficient applications". Trends Biotechnol. 22: 253–260. doi:10.1016/j.tibtech.2004.02.011. PMID 15109812.
^ Rychlik W, Spencer WJ, Rhoads RE (1990). "Optimization of the annealing temperature for DNA amplification in vitro". Nucl Acids Res 18: 6409–6412. doi:10.1093/nar/18.21.6409. PMID 2243783.
^ a b D.J. Sharkey, E.R. Scalice, K.G. Christy Jr., S.M. Atwood, and J.L. Daiss (1994). "Antibodies as Thermolabile Switches: High Temperature Triggering for the Polymerase Chain Reaction". Bio/Technology 12: 506–509. doi:10.1038/nbt0594-506.
^ a b Chien A, Edgar DB, Trela JM (1976). "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus". J. Bacteriol 174: 1550–1557. PMID 8432.
^ a b Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abramson RD, Gelfand DH (1993). "High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity". PCR Methods Appl. 2: 275–287. PMID 8324500.
^ PCR from problematic templates. Focus 22:1 p.10 (2000).
^ Helpful tips for PCR. Focus 22:1 p.12 (2000).
^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes", in Kieleczawa J: DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett, pp. 241-257. ISBN 0-7637338-3-0.
^ Chemical Synthesis, Sequencing, and Amplification of DNA (class notes on MBB/BIO 343). Arizona State University. Retrieved on 2007-10-29.
^ Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, and Markham AF (1989). "Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS)". Nucleic Acids Research 17 (7): 2503–2516. doi:10.1093/nar/17.7.2503. PMID 2785681.
^ Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides". Gene 164: 49–53. doi:10.1016/0378-1119(95)00511-4. PMID 7590320.
^ Innis MA, Myambo KB, Gelfand DH, Brow MA. (1988). "DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA". Proc Natl Acad Sci USA 85: 9436–4940. doi:10.1073/pnas.85.24.9436. PMID 3200828.
^ Pierce KE and Wangh LJ (2007). "Linear-after-the-exponential polymerase chain reaction and allied technologies Real-time detection strategies for rapid, reliable diagnosis from single cells". Methods Mol Med. 132: 65–85. doi:10.1007/978-1-59745-298-4_7. PMID 17876077.
^ Myriam Vincent, Yan Xu and Huimin Kong (2004). "Helicase-dependent isothermal DNA amplification". EMBO reports 5 (8): 795–800. doi:10.1038/sj.embor.7400200.
^ Q. Chou, M. Russell, D.E. Birch, J. Raymond and W. Bloch (1992). "Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications". Nucleic Acids Research 20: 1717–1723. doi:10.1093/nar/20.7.1717.
^ E. Zietkiewicz, A. Rafalski, and D. Labuda (1994). "Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification". Genomics 20 (2): 176–83. doi:10.1006/geno.1994.1151.
^ Ochman H, Gerber AS, Hartl DL (1988). "Genetic applications of an inverse polymerase chain reaction". Genetics 120: 621–623. PMID 2852134.
^ Mueller PR, Wold B (1988). "In vivo footprinting of a muscle specific enhancer by ligation mediated PCR". Science 246: 780–786. doi:10.1126/science.2814500. PMID 2814500.
^ Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB (1996). "Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands". Proc Natl Acad Sci U S A 93 (13): 9821–9826. doi:10.1073/pnas.93.18.9821. PMID 8790415.
^ Isenbarger TA, Finney M, Ríos-Velázquez C, Handelsman J, Ruvkun G (2008). "Miniprimer PCR, a new lens for viewing the microbial world". Applied and Environmental Microbiology 74: 840–9. doi:10.1128/AEM.01933-07. PMID 18083877.
^ Khan Z, Poetter K, Park DJ (2008). "Enhanced solid phase PCR: mechanisms to increase priming by solid support primers". Analytical Biochemistry 375: 391–393. PMID 18267099.
^ Y.G. Liu and R. F. Whittier (1995). "Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking". Genomics 25 (3): 674–81. doi:10.1016/0888-7543(95)80010-J.
^ Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991). "'Touchdown' PCR to circumvent spurious priming during gene amplification". Nucl Acids Res 19: 4008. doi:10.1093/nar/19.14.4008. PMID 1861999.
^ David, F.Turlotte, E., (1998). "An Isothermal Amplification Method". C.R.Acad. Sci Paris, Life Science 321 (1): 909–914.
^ Fabrice David (September-October 2002). Utiliser les propriétés topologiques de l’ADN: une nouvelle arme contre les agents pathogènes. Fusion.(in French)
^ Myrick KV, Gelbart WM (2002). "Universal Fast Walking for direct and versatile determination of flanking sequence". Gene 284: 125–131. doi:10.1016/S0378-1119(02)00384-0. PMID 11891053.
^ Park DJ Electronic Journal of Biotechnology (online). 15 August 2005, vol. 8, no. 2
^ Park DJ (2005). "A new 5' terminal murine GAPDH exon identified using 5'RACE LaNe". Molecular Biotechnology 29: 39–46. doi:10.1385/MB:29:1:39. PMID 15668518.
^ Park DJ (2004). "3'RACE LaNe: a simple and rapid fully nested PCR method to determine 3'-terminal cDNA sequence". Biotechniques 36: 586–588,590. PMID 15088375.
^ Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG (1971). "Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases". J. Mol. Biol. 56: 341–361. doi:10.1016/0022-2836(71)90469-4.
^ Rabinow, Paul (1996). Making PCR: A Story of Biotechnology. Chicago: University of Chicago Press. ISBN 0-226-70146-8.
^ Mullis, Kary (1998). Dancing Naked in the Mind Field. New York: Pantheon Books. ISBN 0-679-44255-3.
^ Mullis, Kary (1990). "The unusual origin of the polymerase chain reaction". Scientific American 262 (4): 56–61, 64–5.
^ Advice on How to Survive the Taq Wars ¶2: GEN Genetic Engineering News Biobusiness Channel: Article. May 1 2006 (Vol. 26, No. 9).
External links
Wikimedia Commons has media related to:
Polymerase chain reaction
PCR at Home - Amateur Scientist article in the July 2000 issue of Scientific American on performing PCRs with low-cost household materials.
US Patent for PCR
Narrated animation and step-through animation of PCR - From the educational multimedia company Sumanas. Adobe Flash required.
Step-through animation of PCR - From Cold Spring Harbor's Dolan DNA Learning Center. Adobe Flash required.
"PCR" redirects here. For other uses, see PCR (disambiguation).
A strip of eight PCR tubes, each containing a 100μl reaction.
The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
Developed in 1983 by Kary Mullis,[1] PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.[2][3] These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis won the Nobel Prize in Chemistry for his work on PCR.[4]
Contents
1 PCR principles and procedure
1.1 Procedure
2 PCR stages
2.1 PCR optimization
3 Application of PCR
3.1 Isolation of genomic DNA
3.2 Amplification and quantitation of DNA
3.3 PCR in diagnosis of diseases
4 Variations on the basic PCR technique
5 History
5.1 Patent wars
6 References
7 External links
PCR principles and procedure
PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.[5]
A basic PCR set up requires several components and reagents.[6] These components include:
DNA template that contains the DNA region (target) to be amplified.
Two primers, which are complementary to the DNA regions at the 5' (five prime) or 3' (three prime) ends of the DNA region.
A DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C.
Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis[7]
Monovalent cation potassium ions.
The PCR is commonly carried out in a reaction volume of 20-150 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.
Procedure
Schematic drawing of the PCR cycle. (1) Denaturing at 94-96°C. (2) Annealing at ~65°C (3) Elongation at 72°C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses.
The PCR usually consists of a series of 20 to 40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps (Fig. 2). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.[8]
Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.[9]
Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C,[10][11] and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.
Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.
To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products.
PCR stages
The PCR process can be divided into three stages:
Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very specific and precise.[citation needed]
Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.
Plateau: No more product accumulates due to exhaustion of reagents and enzyme.
PCR optimization
Main article: PCR optimization
In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.[12][13] Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.[6] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA.
Application of PCR
Isolation of genomic DNA
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.
Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constructs[14]. PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.
Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing. This technique may also be used to determine evolutionary relationships among organisms.
Amplification and quantitation of DNA
Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.[15]
Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample – a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.
See also Use of DNA in forensic entomology
PCR in diagnosis of diseases
PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely.[citation needed] PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.[citation needed]
PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.[citation needed]
Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below).
Variations on the basic PCR technique
Main article: Variants of PCR
Allele-specific PCR: This diagnostic or cloning technique is used to identify or utilize single-nucleotide polymorphisms (SNPs) (single base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.[16] See SNP genotyping for more information.
Assembly PCR or Polymerase Cycling Assembly (PCA): Assembly PCR is the artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final long DNA product.[17]
Asymmetric PCR: Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.[18] A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Melting temperatureTm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.[19]
Helicase-dependent amplification: This technique is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.[20]
Hot-start PCR: This is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase.[21] Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody[9] or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
Intersequence-specific (ISSR) PCR: a PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.[22]
Inverse PCR: a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.[23]
Ligation-mediated PCR: This method uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.[24]
Methylation-specific PCR (MSP): The MSP method was developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine,[25] and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
Miniprimer PCR: Miniprimer PCR uses a novel thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides, instead of the approximately 20 nucleotides required by Taq. This method permits PCR targeting smaller primer binding regions, and is particularly useful to amplify unknown, but conserved, DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene. 16S rRNA miniprimer PCR was used to characterize a microbial mat community growing in an extreme environment, a hypersaline pond in Puerto Rico. In that study, deeply divergent sequences were discovered with high frequency and included representatives that defined two new division-level taxa, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity.[26] By enlarging the "sequence space" that may be queried by PCR primers, this technique may enable novel PCR strategies that are not possible within the limits of primer design imposed by Taq and other commonly used enzymes.
Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).
Multiplex-PCR: The use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
Overlap-extension PCR: is a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.
Quantitative PCR (Q-PCR): is used to measure the quantity of a PCR product (preferably real-time). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR commonly refers to reverse transcription PCR (see below), which is often used in conjunction with Q-PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.
RT-PCR: (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method, named RACE-PCR, short for Rapid Amplification of cDNA Ends.
Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (the only primers present are covalently linked to solid support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR[27] (where conventional Solid Phase PCR can be improved by employing high Tm solid support primer with application of a thermal 'step' to favour solid support priming).
TAIL-PCR: Thermal asymmetric interlaced PCR is used to isolate unknown sequence flanking a known sequence. Within the known sequence TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.[28]
Touchdown PCR: a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5˚C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5˚C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.[29]
PAN-AC: This method uses isothermal conditions for amplification, and may be used in living cells.[30][31]
Universal Fast Walking: this method allows genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer - which can lead to artefactual 'noise') [32] by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends) [33], 5'RACE LaNe [34] and 3'RACE LaNe [35].
History
Main article: History of polymerase chain reaction
A 1971 paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro.[36] However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.[37]
At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90°C (>195°F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures.[2] So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.
A 1976 discovery of Taq polymerase a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F)) environments[10] paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation,[11] thus obviating the need to add new DNA polymerase after each cycle[3]. This allowed an automated thermocycler-based process for DNA amplification.
At the time he developed PCR in 1983, Mullis was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car.[38] He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase.
In Scientific American, Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."[39] He was awarded the Nobel Prize in Chemistry in 1993 for his invention,[4] seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis' work, and whether he had been the sole inventor of the PCR principle. (see main article: Kary Mullis)
Patent wars
The PCR technique was patented by Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.
A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega. The legal arguments have extended beyond the life of the original PCR and Taq polymerase patents, which expired on March 28, 2005[40]
References
^ Bartlett & Stirling (2003)—A Short History of the Polymerase Chain Reaction. In: Methods Mol Biol. 226:3-6
^ a b Saiki, RK; Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (Dec 20 1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science 230 (4732): 1350–4. doi:10.1126/science.2999980. PMID 2999980.
^ a b Saiki, RK; Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science 239: 487–91. doi:10.1126/science.2448875. PMID 2448875.
^ a b Karry Mullis Nobel Lecture, December 8, 1993
^ Cheng S, Fockler C, Barnes WM, Higuchi R (1994). "Effective amplification of long targets from cloned inserts and human genomic DNA". Proc Natl Acad Sci. 91: 5695–5699. doi:10.1073/pnas.91.12.5695. PMID 8202550.
^ a b Joseph Sambrook and David W. Russel (2001). Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. ISBN 0-87969-576-5. Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction
^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004). "Recent developments in the optimization of thermostable DNA polymerases for efficient applications". Trends Biotechnol. 22: 253–260. doi:10.1016/j.tibtech.2004.02.011. PMID 15109812.
^ Rychlik W, Spencer WJ, Rhoads RE (1990). "Optimization of the annealing temperature for DNA amplification in vitro". Nucl Acids Res 18: 6409–6412. doi:10.1093/nar/18.21.6409. PMID 2243783.
^ a b D.J. Sharkey, E.R. Scalice, K.G. Christy Jr., S.M. Atwood, and J.L. Daiss (1994). "Antibodies as Thermolabile Switches: High Temperature Triggering for the Polymerase Chain Reaction". Bio/Technology 12: 506–509. doi:10.1038/nbt0594-506.
^ a b Chien A, Edgar DB, Trela JM (1976). "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus". J. Bacteriol 174: 1550–1557. PMID 8432.
^ a b Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abramson RD, Gelfand DH (1993). "High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity". PCR Methods Appl. 2: 275–287. PMID 8324500.
^ PCR from problematic templates. Focus 22:1 p.10 (2000).
^ Helpful tips for PCR. Focus 22:1 p.12 (2000).
^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes", in Kieleczawa J: DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett, pp. 241-257. ISBN 0-7637338-3-0.
^ Chemical Synthesis, Sequencing, and Amplification of DNA (class notes on MBB/BIO 343). Arizona State University. Retrieved on 2007-10-29.
^ Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, and Markham AF (1989). "Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS)". Nucleic Acids Research 17 (7): 2503–2516. doi:10.1093/nar/17.7.2503. PMID 2785681.
^ Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides". Gene 164: 49–53. doi:10.1016/0378-1119(95)00511-4. PMID 7590320.
^ Innis MA, Myambo KB, Gelfand DH, Brow MA. (1988). "DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA". Proc Natl Acad Sci USA 85: 9436–4940. doi:10.1073/pnas.85.24.9436. PMID 3200828.
^ Pierce KE and Wangh LJ (2007). "Linear-after-the-exponential polymerase chain reaction and allied technologies Real-time detection strategies for rapid, reliable diagnosis from single cells". Methods Mol Med. 132: 65–85. doi:10.1007/978-1-59745-298-4_7. PMID 17876077.
^ Myriam Vincent, Yan Xu and Huimin Kong (2004). "Helicase-dependent isothermal DNA amplification". EMBO reports 5 (8): 795–800. doi:10.1038/sj.embor.7400200.
^ Q. Chou, M. Russell, D.E. Birch, J. Raymond and W. Bloch (1992). "Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications". Nucleic Acids Research 20: 1717–1723. doi:10.1093/nar/20.7.1717.
^ E. Zietkiewicz, A. Rafalski, and D. Labuda (1994). "Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification". Genomics 20 (2): 176–83. doi:10.1006/geno.1994.1151.
^ Ochman H, Gerber AS, Hartl DL (1988). "Genetic applications of an inverse polymerase chain reaction". Genetics 120: 621–623. PMID 2852134.
^ Mueller PR, Wold B (1988). "In vivo footprinting of a muscle specific enhancer by ligation mediated PCR". Science 246: 780–786. doi:10.1126/science.2814500. PMID 2814500.
^ Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB (1996). "Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands". Proc Natl Acad Sci U S A 93 (13): 9821–9826. doi:10.1073/pnas.93.18.9821. PMID 8790415.
^ Isenbarger TA, Finney M, Ríos-Velázquez C, Handelsman J, Ruvkun G (2008). "Miniprimer PCR, a new lens for viewing the microbial world". Applied and Environmental Microbiology 74: 840–9. doi:10.1128/AEM.01933-07. PMID 18083877.
^ Khan Z, Poetter K, Park DJ (2008). "Enhanced solid phase PCR: mechanisms to increase priming by solid support primers". Analytical Biochemistry 375: 391–393. PMID 18267099.
^ Y.G. Liu and R. F. Whittier (1995). "Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking". Genomics 25 (3): 674–81. doi:10.1016/0888-7543(95)80010-J.
^ Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991). "'Touchdown' PCR to circumvent spurious priming during gene amplification". Nucl Acids Res 19: 4008. doi:10.1093/nar/19.14.4008. PMID 1861999.
^ David, F.Turlotte, E., (1998). "An Isothermal Amplification Method". C.R.Acad. Sci Paris, Life Science 321 (1): 909–914.
^ Fabrice David (September-October 2002). Utiliser les propriétés topologiques de l’ADN: une nouvelle arme contre les agents pathogènes. Fusion.(in French)
^ Myrick KV, Gelbart WM (2002). "Universal Fast Walking for direct and versatile determination of flanking sequence". Gene 284: 125–131. doi:10.1016/S0378-1119(02)00384-0. PMID 11891053.
^ Park DJ Electronic Journal of Biotechnology (online). 15 August 2005, vol. 8, no. 2
^ Park DJ (2005). "A new 5' terminal murine GAPDH exon identified using 5'RACE LaNe". Molecular Biotechnology 29: 39–46. doi:10.1385/MB:29:1:39. PMID 15668518.
^ Park DJ (2004). "3'RACE LaNe: a simple and rapid fully nested PCR method to determine 3'-terminal cDNA sequence". Biotechniques 36: 586–588,590. PMID 15088375.
^ Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG (1971). "Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases". J. Mol. Biol. 56: 341–361. doi:10.1016/0022-2836(71)90469-4.
^ Rabinow, Paul (1996). Making PCR: A Story of Biotechnology. Chicago: University of Chicago Press. ISBN 0-226-70146-8.
^ Mullis, Kary (1998). Dancing Naked in the Mind Field. New York: Pantheon Books. ISBN 0-679-44255-3.
^ Mullis, Kary (1990). "The unusual origin of the polymerase chain reaction". Scientific American 262 (4): 56–61, 64–5.
^ Advice on How to Survive the Taq Wars ¶2: GEN Genetic Engineering News Biobusiness Channel: Article. May 1 2006 (Vol. 26, No. 9).
External links
Wikimedia Commons has media related to:
Polymerase chain reaction
PCR at Home - Amateur Scientist article in the July 2000 issue of Scientific American on performing PCRs with low-cost household materials.
US Patent for PCR
Narrated animation and step-through animation of PCR - From the educational multimedia company Sumanas. Adobe Flash required.
Step-through animation of PCR - From Cold Spring Harbor's Dolan DNA Learning Center. Adobe Flash required.
PCR Animations
DNALC: PCR Animation
An animation explaining how the Polymerase Chain Reaction (PCR) works, from the Dolan DNA learning center, Cold Spring Harbor Laboratory, USA.www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html
Polymerase Chain Reaction
RESOURCES. Biology Animation Library. Biology Animation Library ... Polymerase chain reaction (PCR) enables researchers to produce millions of copies of a ...www.dnalc.org/ddnalc/resources/pcr.html
PCR Animation
The PCR depends on the ability to alternately denature (melt) double-stranded DNA molecules and renature (anneal) complementary single strands in a ...www.maxanim.com/genetics/PCR/PCR.htm
PCR animation
highered.mcgraw-hill.com/olc/dl/120078/micro15.swf
Polymerase Chain Reaction
This animation is also available in iPod/iTunes format (.m4v). To download the movie, first review the LICENSE AGREEMENT and click on the link provided. ...www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html
PCR Animation
Animations from Chapter 5. Polymerase Chain Reaction. Close Window.spine.rutgers.edu/cellbio/flash/pcr.htm
PCR animated
Animation illustrating the principle of PCR, from the University of Ghent, Belgium.users.ugent.be/~avierstr/principles/pcrani.html
External Links PCR Animation
The Wittwer DNA Lab in the Pathology Department, University of Utah, Salt Lake City, Utah.dna.utah.edu/PCR_Animation_Links.htm
RT-PCR Methodology
This page is designed to help students understand how RT-PCR works, but it assumes an understanding of PCR. The animation is interactive and requires the ...www.bio.davidson.edu/Courses/immunology/Flash/RT_PCR.html
PCR Animation
PCR. [ Biol 207 ] [ Bell ] [ Newsgroup ] [ CSU Chico ] [ Library ]. Copyright by: Jeff Bell Last Update: Friday, May 15, 1998.www.csuchico.edu/~jbell/Biol207/animations/pcr.html
Polymerase Chain Reaction
Back to PCR Lab.faculty.plattsburgh.edu/donald.slish/PCRmov.html
pcr animationDNA sample DNA segment of interest DNA Polymerase Chain Reaction (PCR) STEP-THROUGH NARRATED DNA sample DNA segment of interest DNA Polymerase Chain ...pcr.az/images/rolik2.swf
Twisted Ladder Media - PCR Animation Demo
This animation shows a brief clip from our PCR animation at a reduced size. The animation will start automatically. Make sure your sound is on. ...www.ornl.gov/hgmis/publicat/twistedladder/pcrdemo.html
Concepts in Biochemistry - Interactive Animations
PCR enables researchers to amplify extremely small samples of DNA, ... PCR is now a common laboratory procedure, and because of its broad range of ...www.wiley.com/legacy/college/boyer/0470003790/animations/pcr/pcr.htm
Genetic Origins
T H E O R Y. This experiment examines PV92, a human-specific Alu insertion on chromosome 16. The PV92 genetic system has only two alleles indicating the ...www.geneticorigins.org/pv92/aluframeset.htm
The cycles of the polymerase chain reaction (PCR), 3D animation ...
Manipulation>Techniques>amplifying>PCR animation Polymerase chain reaction (PCR) is a process where many copies of a specific piece of DNA can be made. ...www.dnai.org/text/582_the_cycles_of_the_polymerase_chain_reaction_pcr_3d_animation_with_no_audio.html
Scanelis - PCR animation, PCR principle
Real-time PCR animation: PCR and Real-time PCR principle.www.scanelis.com/webpages.aspx?rID=679
Biology animations: advantages of PCR animation
Apr 7, 2008 ... biology animation, mitosis animation, meiosis animation, cell animation, cancer animation ... advantages of PCR animation ...biology-animations.blogspot.com/2008/04/advantages-of-pcr-animation.html
Real time pcr animation - Life Science Products - Accurate Biogene ...
Life science products, real time pcr animation and buyer's guide at Accurate Biogene. Search for antibodies, microarrays, immunochemicals, proteins, ...www.accuratebiogene.com/search/real-time-pcr-animation.html
Animations on PCR
The mode of action of SCORPION primers in real time PCR. The mode of action of Taqman primers in real time PCR. Animation of PCR. Animation of PCR. ...www.clinical-virology.org/anim/anim_pcr.html
Real Time PCR Tutorial
The above figures may be found in an animated PowerPoint presentation here ... Now let us turn to real time PCR and, first, to why it was developed ...pathmicro.med.sc.edu/pcr/realtime-home.htm
YouTube - Real time PCR cycle
Real time PCR cycle. Hello, you either have JavaScript turned ...www.youtube.com/watch?v=8-dsnlNsCao
An animation explaining how the Polymerase Chain Reaction (PCR) works, from the Dolan DNA learning center, Cold Spring Harbor Laboratory, USA.www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html
Polymerase Chain Reaction
RESOURCES. Biology Animation Library. Biology Animation Library ... Polymerase chain reaction (PCR) enables researchers to produce millions of copies of a ...www.dnalc.org/ddnalc/resources/pcr.html
PCR Animation
The PCR depends on the ability to alternately denature (melt) double-stranded DNA molecules and renature (anneal) complementary single strands in a ...www.maxanim.com/genetics/PCR/PCR.htm
PCR animation
highered.mcgraw-hill.com/olc/dl/120078/micro15.swf
Polymerase Chain Reaction
This animation is also available in iPod/iTunes format (.m4v). To download the movie, first review the LICENSE AGREEMENT and click on the link provided. ...www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html
PCR Animation
Animations from Chapter 5. Polymerase Chain Reaction. Close Window.spine.rutgers.edu/cellbio/flash/pcr.htm
PCR animated
Animation illustrating the principle of PCR, from the University of Ghent, Belgium.users.ugent.be/~avierstr/principles/pcrani.html
External Links PCR Animation
The Wittwer DNA Lab in the Pathology Department, University of Utah, Salt Lake City, Utah.dna.utah.edu/PCR_Animation_Links.htm
RT-PCR Methodology
This page is designed to help students understand how RT-PCR works, but it assumes an understanding of PCR. The animation is interactive and requires the ...www.bio.davidson.edu/Courses/immunology/Flash/RT_PCR.html
PCR Animation
PCR. [ Biol 207 ] [ Bell ] [ Newsgroup ] [ CSU Chico ] [ Library ]. Copyright by: Jeff Bell Last Update: Friday, May 15, 1998.www.csuchico.edu/~jbell/Biol207/animations/pcr.html
Polymerase Chain Reaction
Back to PCR Lab.faculty.plattsburgh.edu/donald.slish/PCRmov.html
pcr animationDNA sample DNA segment of interest DNA Polymerase Chain Reaction (PCR) STEP-THROUGH NARRATED DNA sample DNA segment of interest DNA Polymerase Chain ...pcr.az/images/rolik2.swf
Twisted Ladder Media - PCR Animation Demo
This animation shows a brief clip from our PCR animation at a reduced size. The animation will start automatically. Make sure your sound is on. ...www.ornl.gov/hgmis/publicat/twistedladder/pcrdemo.html
Concepts in Biochemistry - Interactive Animations
PCR enables researchers to amplify extremely small samples of DNA, ... PCR is now a common laboratory procedure, and because of its broad range of ...www.wiley.com/legacy/college/boyer/0470003790/animations/pcr/pcr.htm
Genetic Origins
T H E O R Y. This experiment examines PV92, a human-specific Alu insertion on chromosome 16. The PV92 genetic system has only two alleles indicating the ...www.geneticorigins.org/pv92/aluframeset.htm
The cycles of the polymerase chain reaction (PCR), 3D animation ...
Manipulation>Techniques>amplifying>PCR animation Polymerase chain reaction (PCR) is a process where many copies of a specific piece of DNA can be made. ...www.dnai.org/text/582_the_cycles_of_the_polymerase_chain_reaction_pcr_3d_animation_with_no_audio.html
Scanelis - PCR animation, PCR principle
Real-time PCR animation: PCR and Real-time PCR principle.www.scanelis.com/webpages.aspx?rID=679
Biology animations: advantages of PCR animation
Apr 7, 2008 ... biology animation, mitosis animation, meiosis animation, cell animation, cancer animation ... advantages of PCR animation ...biology-animations.blogspot.com/2008/04/advantages-of-pcr-animation.html
Real time pcr animation - Life Science Products - Accurate Biogene ...
Life science products, real time pcr animation and buyer's guide at Accurate Biogene. Search for antibodies, microarrays, immunochemicals, proteins, ...www.accuratebiogene.com/search/real-time-pcr-animation.html
Animations on PCR
The mode of action of SCORPION primers in real time PCR. The mode of action of Taqman primers in real time PCR. Animation of PCR. Animation of PCR. ...www.clinical-virology.org/anim/anim_pcr.html
Real Time PCR Tutorial
The above figures may be found in an animated PowerPoint presentation here ... Now let us turn to real time PCR and, first, to why it was developed ...pathmicro.med.sc.edu/pcr/realtime-home.htm
YouTube - Real time PCR cycle
Real time PCR cycle. Hello, you either have JavaScript turned ...www.youtube.com/watch?v=8-dsnlNsCao
PCR, RT-PCR, and Real Time PCR Tutorials
PCR, RT PCR and Real Time PCR Tutorials
Real Time PCR Tutorial
TUTORIAL A Flash tutorial on PCR is here, This type of agarose gel-based analysis of cDNA products of reverse transcriptase-PCR does not allow accurate ...pathmicro.med.sc.edu/pcr/realtime-home.htm
Real Time PCR Tutorial
REAL TIME PCR TUTORIAL. HTML version prepared by Dr Richard Hunt and Srilata Kukunuri, HTML version This HTML presentation contains more information and ...pathmicro.med.sc.edu/pcr/pcr-home.htm
Fast PCR Tutorial
You need Flash Player Version 8 to view the Tutorial. Please follow the link below to update Thanks!www.biocompare.com/tutorials/fast_pcr/
Roche-HIV - PCR Tutorial
DENATURATION The aim of the Polymerase Chain Reaction (PCR) is to identify a particular sequence of DNA and produce many copies of it, until there are so ...www.roche-hiv.com/portal/eipf/pb/hiv/Roche-HIV/pcrtutorial1
DNALC: PCR Animation
An animation explaining how the Polymerase Chain Reaction (PCR) works, from the Dolan DNA learning center, Cold Spring Harbor Laboratory, USA.www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html
YouTube - LinkedIn-PCR Tutorial
Grow your LinkedIn network by leveraging your warm contacts ...www.youtube.com/watch?v=qEHa-8Odegg
Real-Time PCR Vs. Traditional PCRReal-Time PCR Vs. Traditional PCR. Description. This tutorial will discuss the evolution of traditional PCR methods towards. the use of Real-Time chemistry ...www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf
Polymerase Chain Reaction
SOURCE: © 2006 Sumanas, Inc. KEYWORDS: Polymerase chain reaction, DNA amplification, Taq polymerase, genomics This animation is also available in ...www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html
PCR tutorial : Index
Tutorial for Principal Component Regression. ... PCR Tutorial ... Click on a picture to chose the way you will browse this tutorial . ...minf.vub.ac.be/~fabi/calibration/multi/pcr/
LinkedIn-PCR Tutorial on Technorati
LinkedIn-PCR Tutorial. http://youtube.com/watch?v=qEHa-8Odegg. Grow your LinkedIn network by leveraging your warm contacts in PCR! ...technorati.com/videos/youtube.com%2Fwatch%3Fv%3DqEHa-8Odegg
tutorial - PCR primers
Sockeye can help you predict primers using the programs Primer3 and e-PCR. This How-To document will guide you through the steps to perform a primer ...coast.bcgsc.bc.ca:8080/sockeyehelp/1.2/howto/pcr_primers.
Real-Time PCR Vs. Traditional PCR
Real-Time PCR Vs. Traditional PCR. Description. This tutorial will discuss the evolution of traditional PCR methods towards. the use of Real-Time chemistry ...www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf
PCR tutorial for Absolute Beginners
In this short PCR tutorial, I will attempt to give a basics-only knowledge of the reaction, and how to make it work. I will talk about the theory of PCR, ...www.r9corporation.fsnet.co.uk/Methods/PCRtutorialf.htm
Real-Time PCR Tutorial
Quantitative Real-Time PCR, a Practical Tutorial by Joshua Gray, Pharmacology & Toxicology, Rutgers University, Piscataway, NJ. Introduction ...www.rci.rutgers.edu/~gray/RealtimePCR/realmain.htm
Real-time PCR - A Tutorial by Joshua Gray, Ph.D.
Quantitative Real-time PCR, a Tutorial by Joshua Gray, Ph.D. - Assistant Professor, Rutgers University. PCR-based methods for the detection of changes in ...joshuapgray.com/RealtimePCR/realindex.htm
Dr. Chin-Fu Chen's Lab - Real time RT-PCR tutorial website
Real time PCR articles The following is a good tutorial website for real time RT-PCR. Please give it a read before designing and conducting your experiment. ...www.chenlab.clemson.edu/article.php?story=20060217084512839
Real Time PCR Tutorial - Resource Description
Real Time PCR Tutorial. http://pathmicro.med.sc.edu/pcr/realtime-home.htm. This site has a real time pcr tutorial, movie, and PowerPoint presentation. ...www.library.kent.edu/resource.php?id=2269
Biocompare - Fast PCR Tutorial Text
Many researchers suppose that fast PCR can be achieved only by using specialized thermal cyclers with fast ramping rates. In this tutorial, we demonstrate ...www.biocompare.com/tutorials/fast_pcr/Fast_PCR_Tutorial.asp
Real Time PCR Tutorial
TUTORIAL A Flash tutorial on PCR is here, This type of agarose gel-based analysis of cDNA products of reverse transcriptase-PCR does not allow accurate ...pathmicro.med.sc.edu/pcr/realtime-home.htm
Real Time PCR Tutorial
REAL TIME PCR TUTORIAL. HTML version prepared by Dr Richard Hunt and Srilata Kukunuri, HTML version This HTML presentation contains more information and ...pathmicro.med.sc.edu/pcr/pcr-home.htm
Fast PCR Tutorial
You need Flash Player Version 8 to view the Tutorial. Please follow the link below to update Thanks!www.biocompare.com/tutorials/fast_pcr/
Roche-HIV - PCR Tutorial
DENATURATION The aim of the Polymerase Chain Reaction (PCR) is to identify a particular sequence of DNA and produce many copies of it, until there are so ...www.roche-hiv.com/portal/eipf/pb/hiv/Roche-HIV/pcrtutorial1
DNALC: PCR Animation
An animation explaining how the Polymerase Chain Reaction (PCR) works, from the Dolan DNA learning center, Cold Spring Harbor Laboratory, USA.www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html
YouTube - LinkedIn-PCR Tutorial
Grow your LinkedIn network by leveraging your warm contacts ...www.youtube.com/watch?v=qEHa-8Odegg
Real-Time PCR Vs. Traditional PCRReal-Time PCR Vs. Traditional PCR. Description. This tutorial will discuss the evolution of traditional PCR methods towards. the use of Real-Time chemistry ...www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf
Polymerase Chain Reaction
SOURCE: © 2006 Sumanas, Inc. KEYWORDS: Polymerase chain reaction, DNA amplification, Taq polymerase, genomics This animation is also available in ...www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html
PCR tutorial : Index
Tutorial for Principal Component Regression. ... PCR Tutorial ... Click on a picture to chose the way you will browse this tutorial . ...minf.vub.ac.be/~fabi/calibration/multi/pcr/
LinkedIn-PCR Tutorial on Technorati
LinkedIn-PCR Tutorial. http://youtube.com/watch?v=qEHa-8Odegg. Grow your LinkedIn network by leveraging your warm contacts in PCR! ...technorati.com/videos/youtube.com%2Fwatch%3Fv%3DqEHa-8Odegg
tutorial - PCR primers
Sockeye can help you predict primers using the programs Primer3 and e-PCR. This How-To document will guide you through the steps to perform a primer ...coast.bcgsc.bc.ca:8080/sockeyehelp/1.2/howto/pcr_primers.
Real-Time PCR Vs. Traditional PCR
Real-Time PCR Vs. Traditional PCR. Description. This tutorial will discuss the evolution of traditional PCR methods towards. the use of Real-Time chemistry ...www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf
PCR tutorial for Absolute Beginners
In this short PCR tutorial, I will attempt to give a basics-only knowledge of the reaction, and how to make it work. I will talk about the theory of PCR, ...www.r9corporation.fsnet.co.uk/Methods/PCRtutorialf.htm
Real-Time PCR Tutorial
Quantitative Real-Time PCR, a Practical Tutorial by Joshua Gray, Pharmacology & Toxicology, Rutgers University, Piscataway, NJ. Introduction ...www.rci.rutgers.edu/~gray/RealtimePCR/realmain.htm
Real-time PCR - A Tutorial by Joshua Gray, Ph.D.
Quantitative Real-time PCR, a Tutorial by Joshua Gray, Ph.D. - Assistant Professor, Rutgers University. PCR-based methods for the detection of changes in ...joshuapgray.com/RealtimePCR/realindex.htm
Dr. Chin-Fu Chen's Lab - Real time RT-PCR tutorial website
Real time PCR articles The following is a good tutorial website for real time RT-PCR. Please give it a read before designing and conducting your experiment. ...www.chenlab.clemson.edu/article.php?story=20060217084512839
Real Time PCR Tutorial - Resource Description
Real Time PCR Tutorial. http://pathmicro.med.sc.edu/pcr/realtime-home.htm. This site has a real time pcr tutorial, movie, and PowerPoint presentation. ...www.library.kent.edu/resource.php?id=2269
Biocompare - Fast PCR Tutorial Text
Many researchers suppose that fast PCR can be achieved only by using specialized thermal cyclers with fast ramping rates. In this tutorial, we demonstrate ...www.biocompare.com/tutorials/fast_pcr/Fast_PCR_Tutorial.asp
Real Time PCR Protocols
Real-time polymerase chain reaction
From Wikipedia, the free encyclopedia
Jump to: navigation, search
In Molecular Biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase chain reaction, is a laboratory technique based on polymerase chain reaction, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample.
The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-stranded DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.
Frequently, real-time polymerase chain reaction is combined with reverse transcription polymerase chain reaction to quantify low abundance messenger RNA (mRNA), enabling a researcher to quantify relative gene expression at a particular time, or in a particular cell or tissue type.
Although real-time quantitative polymerase chain reaction is often marketed as RT-PCR, it should not be confused with reverse transcription polymerase chain reaction, also known as RT-PCR.
Contents
1 Background
2 Real-time PCR using double-stranded DNA dyes
3 Fluorescent reporter probe method
4 Quantitation
5 Applications of real-time polymerase chain reaction
6 References
7 Further reading
8 External links
REAL TIME RT-PCR PROTOCOL 1. PURPOSE This protocol describes how ...REAL TIME RT-PCR PROTOCOL. SOP #: M011. REVISION LEVEL: .1. PAGE: 2 of 5. 4.1.1 The workbench along with the tube racks and pipetmen should all be ...pga.tigr.org/sop/RT-PCR.pdf
RT-PCR: The Basics
Advantages of real-time RT-PCR; methods, chemistries, ... SYBR Green is the most economical choice for real-time PCR product detection. ...www.ambion.com/techlib/basics/rtpcr/index.html
Real Time PCR
Mar 24, 2008 ... Troubleshooting Real-time PCR Efficiency above 100%, Google, Page 1 ... real time pcr AND troubleshooting AND low fluorescence, Google ...www.scribd.com/doc/2347651/Real-Time-PCR
Real-time quantitative RT-PCR (Taqman)PCR protocol (multiplex reaction). Before the PCR can be performed on the .... e) Real-time quantitative PCR. Once each of the steps a-d have been carried ...www.diabetesgenome.org/docs/Patti_Lab_Real-Time_qPCR_pr.pdf
Real Time PCR
Fairly recently, a new method of PCR quantification has been invented. This is called “real-time PCR” because it allows the scientist to actually view the ...www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Pierce/realtimepcr.htm
Real Time PCR Protocol
Protocol for Real-Time PCR ... You may need to modify this protocol if you use different reagents or instruments for real-time PCR. ...pga.mgh.harvard.edu/primerbank/protocol.html
Protocol for Real-Time RT-PCRTroubleshooting. Here I listed a few major causes for real-time PCR failures. Please read the PrimerBank Help page for more details. ...pga.mgh.harvard.edu/primerbank/PCR_protocol.doc
Protocol for Real-Time RT-PCRAnalyze the real-time PCR result with the SDS 7000 software. Check to see if there is any bimodal dissociation curve or abnormal amplification plot.http://pga.mgh.harvard.edu/primerbank/protocol.html
Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways.
Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Angie L. Bookout and David J. Mangelsdorf [env] ...www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1402222
GMO specific real-time PCR systemGMO specific real-time PCR system. Protocol for event-specific quantitation. of Bt11 in maize. Method development:. National Veterinary Institute (Norway) ...gmo-crl.jrc.it/summaries/Bt11-protocol.pdf
Real-Time PCR [M.Tevfik DORAK]
See also qPCR tips and troubleshooting at qPCR Troubleshooting Guide by ABgene; Protocol Online qPCR troubleshooting; Ten Most Common Real-Time PCR Pitfalls ...dorakmt.tripod.com/genetics/realtime.html
QUANTITATIVE RT-PCR PROTOCOL (SYBR Green I)Quantitative RT-PCR Protocol (SYBR Green I). 4. QUANTITATIVE REAL-TIME PCR (qRT-PCR). 1. Do qRT-PCR and test the selected primers ...schnablelab.plantgenomics.iastate.edu/docs/resources/protocols/pdf/realtime_RT-PCR.2007.04.01.pdf
Bio-Rad iCycler & iQ Real-Time PCR SystemsiCycler iQ Real-time PCR Detection system. 2) Click on the Library module on the left panel. Select the View Protocol tab. Under Protocol Files, select ...www.cosmobio.co.jp/support/technical/manual_SPA_20060904/manual/BioRadiCyclerSetup.pdf
Multiplex Real-Time PCR.
Multiplex Real-Time PCR. This protocol was adapted from "New Technology for Quantitative PCR," contributed by Wolfgang Kusser, Sandrine Javorschi, ...www.cshprotocols.org/cgi/content/full/2006/1/pdb.prot4113
Real Time PCR Tutorial
There are several methods to quantitate alterations in mRNA levels using real time PCR. Let's look at the standard curve method first. ...pathmicro.med.sc.edu/pcr/realtime-home.htm
Real-Time PCR Vs. Traditional PCR
File Format: PDF/Adobe Acrobat - View as HTMLReal-Time PCR Vs. Traditional PCR. Description. This tutorial will discuss the evolution of traditional PCR methods towards. the use of Real-Time chemistry ...www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf
Applied Biosystems Real-Time PCR Rapid Assay Development GuidelinesThis tutorial will discuss recommended guidelines for designing and running. real-time PCR quantification and SNP Genotyping (Allelic Discrimination) ...docs.appliedbiosystems.com/pebiodocs/04371093.pdf
Biocompare - Tools and Technologies for Real-Time PCR
Tools and Technologies for Real-Time PCR. Biocompare's qPCR Tutorial presents researchers with an overview of real-time qPCR, identifies the advantages and ...www.biocompare.com/pcr/tutorial/qpcr/
Real-time PCR - A Tutorial by Joshua Gray, Ph.D.
Quantitative Real-time PCR, a Tutorial by Joshua Gray, Ph.D. - Assistant Professor, Rutgers University. PCR-based methods for the detection of changes in ...joshuapgray.com/RealtimePCR/realindex.htm
Real-Time PCR
Real-time PCR is also the most commonly used method to monitor the ... In addition to this tutorial, our staff can help you with different aspects of your ...qcom.etsu.edu/mbcf/pcr.htm
Real-Time PCR Tutorial
Quantitative Real-Time PCR, a Practical Tutorial by Joshua Gray, Pharmacology & Toxicology, Rutgers University, Piscataway, NJ. Introduction ...www.rci.rutgers.edu/~gray/RealtimePCR/realmain.htm
Real-time PCR - OpenWetWare
Real-time PCR is a PCR technique used to quantify starting amounts of nucleic acid ... An excellent, detailed Q-PCR tutorial by Margaret and Richard Hunt, ...openwetware.org/wiki/Real-time_PCR
Real-Time PCR Primers - Primer Design Ltd
Expert Real Time PCR gene detection kits, for any target gene sequence “on demand”. ... Real-Time PCR Tutorial (South Carolina University) ...www.primerdesign.co.uk/
Real-Time PCR Primer Design
Real-Time PCR Primer Design. ... This online tool designs real-time PCR (TaqMan) primers for you. Number of Primer/Probe Sets to Return (Limit 20 Sets) ...https://www.genscript.com/ssl-bin/app/primer
Oligo Design/Real-Time PCR Primer
Real-Time PCR or qPCR Primer design tools. ... Real-Time PCR Primer Design (Genscript.com) A free online tool for design real-time PCR primers and probes. ...www.protocol-online.org/prot/Research_Tools/Online_Tools/Oligo_Design/Real-Time_PCR_Primer/
Primer Design
When designing only PCR primer pairs, other general primer design programs ... Before to proceeding with real time PCR, it is necessary to test the primers ...www.uic.edu/depts/rrc/cgf/realtime/primer.html
Real-Time PCR Guidelines for designing primers and probes using ...
Keck Home Page > Affymetrix GeneChip Service > Real-Time PCR > Design Guidelines. Guidelines for designing primers and probes using Primer Express. Primer ...keck.med.yale.edu/affymetrix/rtpcr/design.htm
PREMIER Biosoft :: Design & analysis software for Real Time PCR ...
Primer design tools for PCR, real time PCR & microarrays, dual labeled probe ... Design real time PCR primers, TaqMan® probes and molecular beacons for ...www.premierbiosoft.com/
Real Time PCR Primer Design
Design real time PCR primers and TaqMan primers, SYBR Green primers, for quantitative PCR, rt-PCR using AlleleID primer design software.www.premierbiosoft.com/bacterial-identification/primer_design/primer_design.html
Scanelis - PCR animation, PCR principle
Real-time PCR animation: PCR and Real-time PCR principle. ... Download the movie "Real time PCR" in .mov format. Read this format with Apple Quick Time ...www.scanelis.com/webpages.aspx?rID=679 - 21k
Real Time PCR Tutorial
The above figures may be found in an animated PowerPoint presentation here ... Now let us turn to real time PCR and, first, to why it was developed ...pathmicro.med.sc.edu/pcr/realtime-home.htm - 89k
Library of Crop Technology Lessons
You can also click on the animation icon within the text. ... This lesson explains the principles of real time PCR and its application in plant breeding and ...croptechnology.unl.edu/viewLesson.cgi?LessonID=1057077340
real time pcr animation - Life Science Products - Accurate Biogene ...
Life science products, real time pcr animation and buyer's guide at Accurate Biogene. Search for antibodies, microarrays, immunochemicals, proteins, ...www.accuratebiogene.com/search/real-time-pcr-animation.html -
Animations on PCR
The mode of action of SCORPION primers in real time PCR. The mode of action of Taqman primers in real time PCR. Animation of PCR. Animation of PCR. ...www.clinical-virology.org/anim/anim_pcr.html -
PCR Related Sites
An animation from Cold Spring Harbor Laboratories ... Quantitation of DNA/RNA Using Real-time PCR Detection. Applied Biosystems Quantitative RNA Analysis ...www.uq.edu.au/vdu/PCRlinks.htm
YouTube - Real time PCR cycle
Real time PCR cycle. Hello, you either have JavaScript turned ...www.youtube.com/watch?v=8-dsnlNsCao
External Links PCR Animation
Scanelis, PCR and Real-time PCR (animation). The Health News, PCR (animation - narrated). North Harris College Links, Biology I Animations, ...dna.utah.edu/PCR_Animation_Links.htm
Help with Real Time-PCR
Since Real Time - PCR is likely to become a common diagnostic tool during ... It is described in this simple animation at the bottom of this linked page ...www.mgm.ufl.edu/~gulig/mmid/rt-pcr.htm
Applied Biosystems - Support (Tutorial, Mantenance and troubleshooting)
ABI PRISM® 3700 Instrument -Hardware-Raw Data Troubleshooting Module ... Applied Biosystems 7500/7500 Fast Real-Time PCR Systems (7500 Software v2.0, ...www.appliedbiosystems.com/support/apptech/ - 131k
real time pcr troubleshooting guide-vol 3 by genomeTaqMan® Gene Expression Assays deliver accurate real-time PCR results to validate your ..... PCR Troubleshooting: The Essential Guide. Michael Altshuler ...www.genomeweb.com/pdfs/GenomeTech-RTPCRTechGuide-Vol3.pdf
From Wikipedia, the free encyclopedia
Jump to: navigation, search
In Molecular Biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase chain reaction, is a laboratory technique based on polymerase chain reaction, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample.
The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-stranded DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.
Frequently, real-time polymerase chain reaction is combined with reverse transcription polymerase chain reaction to quantify low abundance messenger RNA (mRNA), enabling a researcher to quantify relative gene expression at a particular time, or in a particular cell or tissue type.
Although real-time quantitative polymerase chain reaction is often marketed as RT-PCR, it should not be confused with reverse transcription polymerase chain reaction, also known as RT-PCR.
Contents
1 Background
2 Real-time PCR using double-stranded DNA dyes
3 Fluorescent reporter probe method
4 Quantitation
5 Applications of real-time polymerase chain reaction
6 References
7 Further reading
8 External links
REAL TIME RT-PCR PROTOCOL 1. PURPOSE This protocol describes how ...REAL TIME RT-PCR PROTOCOL. SOP #: M011. REVISION LEVEL: .1. PAGE: 2 of 5. 4.1.1 The workbench along with the tube racks and pipetmen should all be ...pga.tigr.org/sop/RT-PCR.pdf
RT-PCR: The Basics
Advantages of real-time RT-PCR; methods, chemistries, ... SYBR Green is the most economical choice for real-time PCR product detection. ...www.ambion.com/techlib/basics/rtpcr/index.html
Real Time PCR
Mar 24, 2008 ... Troubleshooting Real-time PCR Efficiency above 100%, Google, Page 1 ... real time pcr AND troubleshooting AND low fluorescence, Google ...www.scribd.com/doc/2347651/Real-Time-PCR
Real-time quantitative RT-PCR (Taqman)PCR protocol (multiplex reaction). Before the PCR can be performed on the .... e) Real-time quantitative PCR. Once each of the steps a-d have been carried ...www.diabetesgenome.org/docs/Patti_Lab_Real-Time_qPCR_pr.pdf
Real Time PCR
Fairly recently, a new method of PCR quantification has been invented. This is called “real-time PCR” because it allows the scientist to actually view the ...www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Pierce/realtimepcr.htm
Real Time PCR Protocol
Protocol for Real-Time PCR ... You may need to modify this protocol if you use different reagents or instruments for real-time PCR. ...pga.mgh.harvard.edu/primerbank/protocol.html
Protocol for Real-Time RT-PCRTroubleshooting. Here I listed a few major causes for real-time PCR failures. Please read the PrimerBank Help page for more details. ...pga.mgh.harvard.edu/primerbank/PCR_protocol.doc
Protocol for Real-Time RT-PCRAnalyze the real-time PCR result with the SDS 7000 software. Check to see if there is any bimodal dissociation curve or abnormal amplification plot.http://pga.mgh.harvard.edu/primerbank/protocol.html
Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways.
Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Angie L. Bookout and David J. Mangelsdorf [env] ...www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1402222
GMO specific real-time PCR systemGMO specific real-time PCR system. Protocol for event-specific quantitation. of Bt11 in maize. Method development:. National Veterinary Institute (Norway) ...gmo-crl.jrc.it/summaries/Bt11-protocol.pdf
Real-Time PCR [M.Tevfik DORAK]
See also qPCR tips and troubleshooting at qPCR Troubleshooting Guide by ABgene; Protocol Online qPCR troubleshooting; Ten Most Common Real-Time PCR Pitfalls ...dorakmt.tripod.com/genetics/realtime.html
QUANTITATIVE RT-PCR PROTOCOL (SYBR Green I)Quantitative RT-PCR Protocol (SYBR Green I). 4. QUANTITATIVE REAL-TIME PCR (qRT-PCR). 1. Do qRT-PCR and test the selected primers ...schnablelab.plantgenomics.iastate.edu/docs/resources/protocols/pdf/realtime_RT-PCR.2007.04.01.pdf
Bio-Rad iCycler & iQ Real-Time PCR SystemsiCycler iQ Real-time PCR Detection system. 2) Click on the Library module on the left panel. Select the View Protocol tab. Under Protocol Files, select ...www.cosmobio.co.jp/support/technical/manual_SPA_20060904/manual/BioRadiCyclerSetup.pdf
Multiplex Real-Time PCR.
Multiplex Real-Time PCR. This protocol was adapted from "New Technology for Quantitative PCR," contributed by Wolfgang Kusser, Sandrine Javorschi, ...www.cshprotocols.org/cgi/content/full/2006/1/pdb.prot4113
Real Time PCR Tutorial
There are several methods to quantitate alterations in mRNA levels using real time PCR. Let's look at the standard curve method first. ...pathmicro.med.sc.edu/pcr/realtime-home.htm
Real-Time PCR Vs. Traditional PCR
File Format: PDF/Adobe Acrobat - View as HTMLReal-Time PCR Vs. Traditional PCR. Description. This tutorial will discuss the evolution of traditional PCR methods towards. the use of Real-Time chemistry ...www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf
Applied Biosystems Real-Time PCR Rapid Assay Development GuidelinesThis tutorial will discuss recommended guidelines for designing and running. real-time PCR quantification and SNP Genotyping (Allelic Discrimination) ...docs.appliedbiosystems.com/pebiodocs/04371093.pdf
Biocompare - Tools and Technologies for Real-Time PCR
Tools and Technologies for Real-Time PCR. Biocompare's qPCR Tutorial presents researchers with an overview of real-time qPCR, identifies the advantages and ...www.biocompare.com/pcr/tutorial/qpcr/
Real-time PCR - A Tutorial by Joshua Gray, Ph.D.
Quantitative Real-time PCR, a Tutorial by Joshua Gray, Ph.D. - Assistant Professor, Rutgers University. PCR-based methods for the detection of changes in ...joshuapgray.com/RealtimePCR/realindex.htm
Real-Time PCR
Real-time PCR is also the most commonly used method to monitor the ... In addition to this tutorial, our staff can help you with different aspects of your ...qcom.etsu.edu/mbcf/pcr.htm
Real-Time PCR Tutorial
Quantitative Real-Time PCR, a Practical Tutorial by Joshua Gray, Pharmacology & Toxicology, Rutgers University, Piscataway, NJ. Introduction ...www.rci.rutgers.edu/~gray/RealtimePCR/realmain.htm
Real-time PCR - OpenWetWare
Real-time PCR is a PCR technique used to quantify starting amounts of nucleic acid ... An excellent, detailed Q-PCR tutorial by Margaret and Richard Hunt, ...openwetware.org/wiki/Real-time_PCR
Real-Time PCR Primers - Primer Design Ltd
Expert Real Time PCR gene detection kits, for any target gene sequence “on demand”. ... Real-Time PCR Tutorial (South Carolina University) ...www.primerdesign.co.uk/
Real-Time PCR Primer Design
Real-Time PCR Primer Design. ... This online tool designs real-time PCR (TaqMan) primers for you. Number of Primer/Probe Sets to Return (Limit 20 Sets) ...https://www.genscript.com/ssl-bin/app/primer
Oligo Design/Real-Time PCR Primer
Real-Time PCR or qPCR Primer design tools. ... Real-Time PCR Primer Design (Genscript.com) A free online tool for design real-time PCR primers and probes. ...www.protocol-online.org/prot/Research_Tools/Online_Tools/Oligo_Design/Real-Time_PCR_Primer/
Primer Design
When designing only PCR primer pairs, other general primer design programs ... Before to proceeding with real time PCR, it is necessary to test the primers ...www.uic.edu/depts/rrc/cgf/realtime/primer.html
Real-Time PCR Guidelines for designing primers and probes using ...
Keck Home Page > Affymetrix GeneChip Service > Real-Time PCR > Design Guidelines. Guidelines for designing primers and probes using Primer Express. Primer ...keck.med.yale.edu/affymetrix/rtpcr/design.htm
PREMIER Biosoft :: Design & analysis software for Real Time PCR ...
Primer design tools for PCR, real time PCR & microarrays, dual labeled probe ... Design real time PCR primers, TaqMan® probes and molecular beacons for ...www.premierbiosoft.com/
Real Time PCR Primer Design
Design real time PCR primers and TaqMan primers, SYBR Green primers, for quantitative PCR, rt-PCR using AlleleID primer design software.www.premierbiosoft.com/bacterial-identification/primer_design/primer_design.html
Scanelis - PCR animation, PCR principle
Real-time PCR animation: PCR and Real-time PCR principle. ... Download the movie "Real time PCR" in .mov format. Read this format with Apple Quick Time ...www.scanelis.com/webpages.aspx?rID=679 - 21k
Real Time PCR Tutorial
The above figures may be found in an animated PowerPoint presentation here ... Now let us turn to real time PCR and, first, to why it was developed ...pathmicro.med.sc.edu/pcr/realtime-home.htm - 89k
Library of Crop Technology Lessons
You can also click on the animation icon within the text. ... This lesson explains the principles of real time PCR and its application in plant breeding and ...croptechnology.unl.edu/viewLesson.cgi?LessonID=1057077340
real time pcr animation - Life Science Products - Accurate Biogene ...
Life science products, real time pcr animation and buyer's guide at Accurate Biogene. Search for antibodies, microarrays, immunochemicals, proteins, ...www.accuratebiogene.com/search/real-time-pcr-animation.html -
Animations on PCR
The mode of action of SCORPION primers in real time PCR. The mode of action of Taqman primers in real time PCR. Animation of PCR. Animation of PCR. ...www.clinical-virology.org/anim/anim_pcr.html -
PCR Related Sites
An animation from Cold Spring Harbor Laboratories ... Quantitation of DNA/RNA Using Real-time PCR Detection. Applied Biosystems Quantitative RNA Analysis ...www.uq.edu.au/vdu/PCRlinks.htm
YouTube - Real time PCR cycle
Real time PCR cycle. Hello, you either have JavaScript turned ...www.youtube.com/watch?v=8-dsnlNsCao
External Links PCR Animation
Scanelis, PCR and Real-time PCR (animation). The Health News, PCR (animation - narrated). North Harris College Links, Biology I Animations, ...dna.utah.edu/PCR_Animation_Links.htm
Help with Real Time-PCR
Since Real Time - PCR is likely to become a common diagnostic tool during ... It is described in this simple animation at the bottom of this linked page ...www.mgm.ufl.edu/~gulig/mmid/rt-pcr.htm
Applied Biosystems - Support (Tutorial, Mantenance and troubleshooting)
ABI PRISM® 3700 Instrument -Hardware-Raw Data Troubleshooting Module ... Applied Biosystems 7500/7500 Fast Real-Time PCR Systems (7500 Software v2.0, ...www.appliedbiosystems.com/support/apptech/ - 131k
real time pcr troubleshooting guide-vol 3 by genomeTaqMan® Gene Expression Assays deliver accurate real-time PCR results to validate your ..... PCR Troubleshooting: The Essential Guide. Michael Altshuler ...www.genomeweb.com/pdfs/GenomeTech-RTPCRTechGuide-Vol3.pdf
REAL TIME PCR Papers
An Introduction to Real-Time PCRN.A. SaundersThe development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a very significant advance. The technology is very flexible and many alternative instruments and fluorescent probe systems have been developed and are currently available. Real-time PCR assays can be completed very rapidly since no manipulations are required post-amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with size analysis on gels.
Real-Time PCR PlatformsM.J. Logan and K.J. EdwardsReal-time PCR continues to have a major impact across many disciplines of the biological sciences and this has been a driver to develop and improve existing instruments. From the first two commercial platforms introduced in the mid 1990s, there is now a choice in excess of a dozen instruments, which continues to increase. Advances include faster thermocycling times, higher throughput, flexibility, expanded optical systems, increased multiplexing and more user-friendly software.
Homogeneous Fluorescent Chemistries for Real-Time PCRM.A. Lee, D.J. Squirrell, D.L. Leslie, and T. BrownThe development of fluorescent methods for a closed tube polymerase chain reaction has greatly simplified the process of quantification. Current approaches use fluorescent probes that interact with the amplification products during the PCR to allow kinetic measurements of product accumulation. These probe methods include generic approaches to DNA quantification such as fluorescent DNA binding dyes.
Performing Real-Time PCRK.J. EdwardsOptimisation of the reagents used to perform PCR is critical for reliable and reproducible results. As with any PCR initial time spent on optimisation of a real-time assay will be beneficial in the long run. Specificity, sensitivity, efficiency and reproducibility are the important criteria to consider when optimising an assay and these can be altered by changes in the primer concentration, probe concentration, cycling conditions and buffer composition. An optimised real-time PCR assay will display no test-to-test variation in the crossing threshold or crossing point and only minimal variation in the amount of fluorescence.
Internal and External Controls for Reagent ValidationM.A. Lee, D.L. Leslie and D.J. SquirrellPCR applications that require a high confidence in the result should be designed to control for the occurrence of false negatives. False negatives can occur from inhibition of one or more of the reaction components by a range of factors. While an external, or batch control is often used, the ideal control is one that is included in the reaction cocktail in a multiplex format. Early approaches used different sized amplicons combined with end-point analysis. Fluorescent homogenous real-time PCR methods have a number of advantages for implementing internal controls.
Quantitative Real-Time PCRN.A. SaundersUnlike classical end-point analysis PCR, real-time PCR provides the data required for quantification of the target nucleic acid. The results can be expressed in absolute terms by reference to external quantified standards or in relative terms compared to another target sequence present within the sample. Absolute quantification requires that the efficiency of the amplification reaction is the same in all samples and in the external quantified standards. Consequently, it is important that the efficiency of the PCR does not vary greatly due to minor differences between samples. Careful optimisation of the PCR conditions is therefore required. The use of probes in quantitative real-time PCR improves its performance and a range of suitable systems is now available.
Analysis of mRNA Expression by Real-Time PCRS.A. Bustin and T. NolanThe last few years have seen the transformation of the fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) from an experimental tool into a mainstream scientific technology. Assays are simple to perform, capable of high throughput, and combine high sensitivity with exquisite specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation and has become the "Gold Standard" for a huge range of applications in basic research, molecular medicine, and biotechnology.
Mutation Detection by Real-Time PCRK.J. Edwards and J.M.J LoganReal-time PCR is ideally suited for analysis of single nucleotide polymorphisms (SNPs) and has been increasingly used for this purpose since the advent of real-time PCR and as whole genome sequences have become available. It requires methods that are rapid, sensitive, specific and inexpensive, and several real-time methods have evolved which fulfil these requirements.
The Quantitative Amplification Refractory Mutation SystemP. Punia and N.A. SaundersThe amplification refractory mutation system (ARMS), which has also been described as allele-specific PCR (ASP) and PCR amplification of specific alleles (PASA), is a PCR-based method of detecting single base mutations. ARMS has been applied successfully to the analysis of a wide range of polymorphisms, germ-line mutations and somatic mutations. The technique has the ability to discriminate low-levels of the mutant sequence in a high background of wild-type DNA. In an ARMS PCR the terminal 3' nucleotide of one of the PCR primers coincides with the target mutation. Most applications of the method rely on 'end-point' analysis, utilising the classic gel-electrophoresis method.
Real-Time NASBAS. Hibbitts and J.D. FoxNASBA is an isothermal nucleic acid amplification method that is particularly suited to detection and quantification of genomic, ribosomal or messenger RNA. The product of NASBA is single-stranded RNA of opposite sense to the original target. The first developed NASBA methods relied on liquid or gel-based probe-hybridisation for post-amplification detection of products. More recently, real-time procedures incorporating amplification and detection in a single step have been reported and applied to a wide range of targets. Thus real-time NASBA has proved to be the basis of sensitive and specific assays for detection, quantification and analysis of RNA (and in one case DNA) targets.
Applications of Real-Time PCR in Clinical MicrobiologyA.D. SailsThe introduction of real-time PCR assays to the clinical microbiology laboratory has led to significant improvements in the diagnosis of infectious disease. There has been an explosion of interest in this technique since its introduction and several hundred reports have been published describing applications in clinical bacteriology, parasitology and virology. There are few areas of clinical microbiology which remain unaffected by this new method. It has been particularly useful to detect slow growing or difficult to grow infectious agents. However, its greatest impact is probably its use for the quantitation of target organisms in samples.
Application of Real-Time PCR to the Diagnosis of Invasive Fungal InfectionN. Isik and N.A. SaundersThe management of invasive fungal infections has been hampered by the inability to make a diagnosis at an early stage of the disease. Molecular diagnosis by PCR appears very promising since fungal DNA can be detected in the blood of infected patients earlier than when using conventional methods. Recently, interest in the diagnosis of invasive fungal infections by real-time PCR has increased. Real-time methods also have quantitative properties and are useful both for initial diagnosis and to assess the response to treatment. Many recent studies have combined serological tests with measurement of fungal DNA by using real-time PCR. Real-time PCR helps early diagnosis and arrangement of treatment protocols for patients with high risk of fungal infection.
General PCR Articles
Endonuclease-Mediated Long PCR and Its Application to Restriction Mapping Curr. Issues Mol. Biol. (1999) 1: 77-88 Chengtao Her and Richard M. WeinshilboumThe polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for PCR have been extended significantly by the development of "long" PCR.
A PCR-based Method for Isolation of Genomic DNA Flanking a Known DNA Sequence Curr. Issues Mol. Biol. (1999) 1: 47-52 Catherine A. Boulter and Dipa NatarajanA simple PCR-based method for the isolation of genomic DNA that lies adjacent to a known DNA sequence.
Universal TA Cloning Curr. Issues Mol. Biol. (2000) 2: 1-7 Ming-Yi Zhou and Celso E. Gomez-SanchezTA cloning is one of the simplest and most efficient methods for the cloning of PCR products.
Analysis of Specific Bacteria from Environmental Samples using a Quantitative Polymerase Chain Reaction Curr. Issues Mol. Biol. (2002) 4: 13-18 Clifford F. Brunk, Jinliang Li and Erik Avaniss-AghajaniThe use of quantitative PCR for measuring bacterial abundance in environmental samples.
PCR Clamping Curr. Issues Mol. Biol. (2000) 2: 27-30 Henrik ØrumAn efficient, PCR based method for the selective amplification of DNA target sequences that differ by a single base pair. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases.
DNA Splicing by Directed Ligation (SDL) Curr. Issues Mol. Biol. (1999) 1: 21-30 DNA Yuri A. BerlinSplicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites.
PCR Family Brochure.book
File Format: PDF/Adobe AcrobatA basic set of PCR (chapter 4) and RT-PCR (chapter 5) protocols, including tips on. how to get the best results with our products. ...www.roche-applied-science.com/PROD_INF/MANUALS/pcr_man/chapter_1.pdf
Real-Time PCR PlatformsM.J. Logan and K.J. EdwardsReal-time PCR continues to have a major impact across many disciplines of the biological sciences and this has been a driver to develop and improve existing instruments. From the first two commercial platforms introduced in the mid 1990s, there is now a choice in excess of a dozen instruments, which continues to increase. Advances include faster thermocycling times, higher throughput, flexibility, expanded optical systems, increased multiplexing and more user-friendly software.
Homogeneous Fluorescent Chemistries for Real-Time PCRM.A. Lee, D.J. Squirrell, D.L. Leslie, and T. BrownThe development of fluorescent methods for a closed tube polymerase chain reaction has greatly simplified the process of quantification. Current approaches use fluorescent probes that interact with the amplification products during the PCR to allow kinetic measurements of product accumulation. These probe methods include generic approaches to DNA quantification such as fluorescent DNA binding dyes.
Performing Real-Time PCRK.J. EdwardsOptimisation of the reagents used to perform PCR is critical for reliable and reproducible results. As with any PCR initial time spent on optimisation of a real-time assay will be beneficial in the long run. Specificity, sensitivity, efficiency and reproducibility are the important criteria to consider when optimising an assay and these can be altered by changes in the primer concentration, probe concentration, cycling conditions and buffer composition. An optimised real-time PCR assay will display no test-to-test variation in the crossing threshold or crossing point and only minimal variation in the amount of fluorescence.
Internal and External Controls for Reagent ValidationM.A. Lee, D.L. Leslie and D.J. SquirrellPCR applications that require a high confidence in the result should be designed to control for the occurrence of false negatives. False negatives can occur from inhibition of one or more of the reaction components by a range of factors. While an external, or batch control is often used, the ideal control is one that is included in the reaction cocktail in a multiplex format. Early approaches used different sized amplicons combined with end-point analysis. Fluorescent homogenous real-time PCR methods have a number of advantages for implementing internal controls.
Quantitative Real-Time PCRN.A. SaundersUnlike classical end-point analysis PCR, real-time PCR provides the data required for quantification of the target nucleic acid. The results can be expressed in absolute terms by reference to external quantified standards or in relative terms compared to another target sequence present within the sample. Absolute quantification requires that the efficiency of the amplification reaction is the same in all samples and in the external quantified standards. Consequently, it is important that the efficiency of the PCR does not vary greatly due to minor differences between samples. Careful optimisation of the PCR conditions is therefore required. The use of probes in quantitative real-time PCR improves its performance and a range of suitable systems is now available.
Analysis of mRNA Expression by Real-Time PCRS.A. Bustin and T. NolanThe last few years have seen the transformation of the fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) from an experimental tool into a mainstream scientific technology. Assays are simple to perform, capable of high throughput, and combine high sensitivity with exquisite specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation and has become the "Gold Standard" for a huge range of applications in basic research, molecular medicine, and biotechnology.
Mutation Detection by Real-Time PCRK.J. Edwards and J.M.J LoganReal-time PCR is ideally suited for analysis of single nucleotide polymorphisms (SNPs) and has been increasingly used for this purpose since the advent of real-time PCR and as whole genome sequences have become available. It requires methods that are rapid, sensitive, specific and inexpensive, and several real-time methods have evolved which fulfil these requirements.
The Quantitative Amplification Refractory Mutation SystemP. Punia and N.A. SaundersThe amplification refractory mutation system (ARMS), which has also been described as allele-specific PCR (ASP) and PCR amplification of specific alleles (PASA), is a PCR-based method of detecting single base mutations. ARMS has been applied successfully to the analysis of a wide range of polymorphisms, germ-line mutations and somatic mutations. The technique has the ability to discriminate low-levels of the mutant sequence in a high background of wild-type DNA. In an ARMS PCR the terminal 3' nucleotide of one of the PCR primers coincides with the target mutation. Most applications of the method rely on 'end-point' analysis, utilising the classic gel-electrophoresis method.
Real-Time NASBAS. Hibbitts and J.D. FoxNASBA is an isothermal nucleic acid amplification method that is particularly suited to detection and quantification of genomic, ribosomal or messenger RNA. The product of NASBA is single-stranded RNA of opposite sense to the original target. The first developed NASBA methods relied on liquid or gel-based probe-hybridisation for post-amplification detection of products. More recently, real-time procedures incorporating amplification and detection in a single step have been reported and applied to a wide range of targets. Thus real-time NASBA has proved to be the basis of sensitive and specific assays for detection, quantification and analysis of RNA (and in one case DNA) targets.
Applications of Real-Time PCR in Clinical MicrobiologyA.D. SailsThe introduction of real-time PCR assays to the clinical microbiology laboratory has led to significant improvements in the diagnosis of infectious disease. There has been an explosion of interest in this technique since its introduction and several hundred reports have been published describing applications in clinical bacteriology, parasitology and virology. There are few areas of clinical microbiology which remain unaffected by this new method. It has been particularly useful to detect slow growing or difficult to grow infectious agents. However, its greatest impact is probably its use for the quantitation of target organisms in samples.
Application of Real-Time PCR to the Diagnosis of Invasive Fungal InfectionN. Isik and N.A. SaundersThe management of invasive fungal infections has been hampered by the inability to make a diagnosis at an early stage of the disease. Molecular diagnosis by PCR appears very promising since fungal DNA can be detected in the blood of infected patients earlier than when using conventional methods. Recently, interest in the diagnosis of invasive fungal infections by real-time PCR has increased. Real-time methods also have quantitative properties and are useful both for initial diagnosis and to assess the response to treatment. Many recent studies have combined serological tests with measurement of fungal DNA by using real-time PCR. Real-time PCR helps early diagnosis and arrangement of treatment protocols for patients with high risk of fungal infection.
General PCR Articles
Endonuclease-Mediated Long PCR and Its Application to Restriction Mapping Curr. Issues Mol. Biol. (1999) 1: 77-88 Chengtao Her and Richard M. WeinshilboumThe polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for PCR have been extended significantly by the development of "long" PCR.
A PCR-based Method for Isolation of Genomic DNA Flanking a Known DNA Sequence Curr. Issues Mol. Biol. (1999) 1: 47-52 Catherine A. Boulter and Dipa NatarajanA simple PCR-based method for the isolation of genomic DNA that lies adjacent to a known DNA sequence.
Universal TA Cloning Curr. Issues Mol. Biol. (2000) 2: 1-7 Ming-Yi Zhou and Celso E. Gomez-SanchezTA cloning is one of the simplest and most efficient methods for the cloning of PCR products.
Analysis of Specific Bacteria from Environmental Samples using a Quantitative Polymerase Chain Reaction Curr. Issues Mol. Biol. (2002) 4: 13-18 Clifford F. Brunk, Jinliang Li and Erik Avaniss-AghajaniThe use of quantitative PCR for measuring bacterial abundance in environmental samples.
PCR Clamping Curr. Issues Mol. Biol. (2000) 2: 27-30 Henrik ØrumAn efficient, PCR based method for the selective amplification of DNA target sequences that differ by a single base pair. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases.
DNA Splicing by Directed Ligation (SDL) Curr. Issues Mol. Biol. (1999) 1: 21-30 DNA Yuri A. BerlinSplicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites.
PCR Family Brochure.book
File Format: PDF/Adobe AcrobatA basic set of PCR (chapter 4) and RT-PCR (chapter 5) protocols, including tips on. how to get the best results with our products. ...www.roche-applied-science.com/PROD_INF/MANUALS/pcr_man/chapter_1.pdf
Variants of PCR (5)
Algorithm application improves lab experiments
CDC - Real-Time Reverse Transcription–Polymerase Chain Reaction …
Chip-based lab cuts PCR time
Cloning a gene (polymerase chain reaction)
Electronic PCR
In silico PCR
Long PCR Protocol
M EDIA and A NIMATION Polymerase Chain Reaction (All Cycles) M …
PCR
PCR (Polymerase Chain Reaction) - HIV: health and medical …
PCR Application Roundup - MacResearch
PCR Box Titration Calculator
PCR Project
PCR Protocols
PCR Protocols Guide
PCR trouble shooting, help, suggestions and advice
Polymerase Chain Reaction
Polymerase Chain Reaction
Polymerase Chain Reaction
Polymerase Chain Reaction (PCR)
Principle of PCR
Quantitative Real-Time Polymerase Chain Reaction
rDNA: Polymerase Chain Reaction (PCR)
Real Time PCR Tutorial
Real-Time PCR [M.Tevfik DORAK]
RT-PCR Methodology
RT-PCR: The Basics
Technical manual of PCR
The Polymerase Chain Reaction (PCR): Cloning DNA in the Test Tube Troubleshooting for PCR and multiplex PCR
CDC - Real-Time Reverse Transcription–Polymerase Chain Reaction …
Chip-based lab cuts PCR time
Cloning a gene (polymerase chain reaction)
Electronic PCR
In silico PCR
Long PCR Protocol
M EDIA and A NIMATION Polymerase Chain Reaction (All Cycles) M …
PCR
PCR (Polymerase Chain Reaction) - HIV: health and medical …
PCR Application Roundup - MacResearch
PCR Box Titration Calculator
PCR Project
PCR Protocols
PCR Protocols Guide
PCR trouble shooting, help, suggestions and advice
Polymerase Chain Reaction
Polymerase Chain Reaction
Polymerase Chain Reaction
Polymerase Chain Reaction (PCR)
Principle of PCR
Quantitative Real-Time Polymerase Chain Reaction
rDNA: Polymerase Chain Reaction (PCR)
Real Time PCR Tutorial
Real-Time PCR [M.Tevfik DORAK]
RT-PCR Methodology
RT-PCR: The Basics
Technical manual of PCR
The Polymerase Chain Reaction (PCR): Cloning DNA in the Test Tube Troubleshooting for PCR and multiplex PCR
RT PCR Protocols
RT-PCR Protocol
RT-PCR protocol. RNA preparation. 1. Grow cells to confluence in a single well of a 6-well plate. 2. Lyse the cells with 1 ml Trizol reagent. ...www.sanger.ac.uk/PostGenomics/genetrap/protocols/RTPCR.pdf
RT-PCR Protocol
RT-PCR Protocol. cDNA preparation: 4-5µL RNA 3µL oligo dT primer 12µL DEPC-treated water Incubate at 70 degrees for 10 minutes, then put on ice. ...preuss.bsd.uchicago.edu/protocols/RT.html
Protocol #3 RT-PCR and PCR
Protocol #3. RT-PCR. Protocol #3. RT-PCR and PCR. Introduction. The RT-PCR kit contains polymerase and all necessary reagents to perform single tube RT and ...www.ucc.ie/ucc/depts/physio/MolPhysiol/MolPhys/Protocols/pdfs/003.pdf
RT-PCR Protocol ain this protocol is thermostable, reverse transcription time is cut in half. The speci-. ficity and sensitivity associated with RT-PCR is also heightened as ...www.mbpinc.com/html/pdf/products/pcr/RT-PCR%20Protocol%20a.pdf
RT PCRRT PCR PROTOCOL: BD BIOSCIENCES. . This protocol is adapted from a BDBiosciences protocol by the Gene Expression Lab. This protocol is for use with BD ...www.ncifcrf.gov/atp/LMT/RT_PCR_Protocol.pdf
Relative Quantitative RT-PCR Protocol.
Relative Quantitative RT-PCR Protocol. Wild-type and experimental plants should be planted and grown in parallel. Wild-type and experimental plants’ RNA ...www.chromatin-consortium.org/docs/rt-pcr_protocol.pdf
Protocol: a highly sensitive RT-PCR Method for Detection and Quantification of mRNAs.
Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and ...www.plantmethods.com/content/3/1/12
QIAGEN OneStep RT-PCR Handbook
This protocol serves as a guideline for one-step RT-PCR. ...... The QIAGEN OneStep RT-PCR Protocol is optimized for amplification of products of up ...www1.qiagen.com/HB/OneStepRTPCR
RT-PCR: Two-Step Protocol Two-step Protocol
RT-PCR: Two-Step Protocol. We will provide both one-step and two-step protocols ... In the one-step protocol, the components of RT and PCR are mixed in a ...ocw.mit.edu/NR/rdonlyres/Biology/7-16Spring-2005/85278F14-478B-469E-8D2E
Atlas Arrays RT-PCR User Manual
User Manual provides an RT-PCR protocol and related information for use .... RT-PCR Protocol continued. 11. The cDNA is now ready for immediate use or ...www.clontech.com/images/pt/PT3270-1.pdf
cDNA Synthesis for RT-PCR Protocol Reagents Procedure
cDNA Synthesis for RT-PCR Protocol. Section of Cancer Genomics, Genetics Branch, NCI. National Institutes of Health. Reagents. 5X First Strand Buffer ...www.riedlab.nci.nih.gov/publications/cDNA%20Synthesis%20for%20RT-PCR.pdf
Ambion: Automation of One-Step RT-PCR Using Cells-to-cDNA II
This automated protocol includes cell lysis, DNase I treatment, and set-up for one-step RT-PCR, that can then be used in gel-based or quantitative ...www.ambion.com/techlib/posters/c2cdna_automation_0204.html
Protocol for Preparation of DNA-free RNA Prior to RT-PCR
Protocol for Preparation of DNA-free RNA Prior to RT-PCR (with DNase I, RNase-free). Add to an RNase-free tube:. RNA, 1 µg. 10X reaction buffer with MgCl2 ...www.fermentas.com/techinfo/modifyingenzymes/protocols/p_prepdnafreerna.htm
Protocol Using QIAGEN OneStep RT-PCR Kit
This protocol serves as a guideline for one-step RT-PCR. .... QIAGEN OneStep RT-PCR. Protocol. Table 2. Thermal cycler conditions. Additional comments ...www.uark.edu/ua/henrylab/Links/biochemgen/QIAGEN%20RT-PCR%20protocol.pdf
rt pcr animation
RT-PCR stands for Reverse Transcription-Polymerase Chain Reaction. ... with primers and Taq Polymerase, and the standard PCR protocol is followed. ...www.bio.davidson.edu/people/kabernd/seminar/2002/method/lowry/RTPCR.htm
PCRboost Quick Reference Protocol
When using PCRboost, there is no change to your PCR or. RT-PCR protocol. Simply substitute PCRboost for water. The following are examples of PCR and RT-PCR ...www.biomatrica.com/media/PCRBoostProtocols.pdf
Two-Step RT-PCR Kit
PROTOCOL A:TWO-STEP RT-PCR. This standard protocol applies to a single ..... RT-PCR is carried out according to the standard protocol with 100 ng total RNA ...www.usbweb.com/proto/78355a.pdf
DNA Contamination in RT-PCR (Technical Bulletin #176)
Ways to get rid of DNA contamination in RNA samples--a frequent cause of concern among investigators performing quantitative RT-PCR.www.ambion.com/techlib/tb/tb_176.html
Reverse Transcriptase-PCR
Reverse Transcriptase-PCR Lazo Lab/Jim Hutchins
http://wheat.pw.usda.gov/~lazo/methods/lazo/revtrpcr.html
RT-PCR of Undifferentiated Cells [Stem Cell Information]
Skip to content. Click to visit the National Institutes of Health Web site · Stem Cell Information. Stem Cell Information. The National Institutes of Health ...stemcells.nih.gov/research/NIHresearch/scunit/RTPCR.asp
RT-PCR protocol. RNA preparation. 1. Grow cells to confluence in a single well of a 6-well plate. 2. Lyse the cells with 1 ml Trizol reagent. ...www.sanger.ac.uk/PostGenomics/genetrap/protocols/RTPCR.pdf
RT-PCR Protocol
RT-PCR Protocol. cDNA preparation: 4-5µL RNA 3µL oligo dT primer 12µL DEPC-treated water Incubate at 70 degrees for 10 minutes, then put on ice. ...preuss.bsd.uchicago.edu/protocols/RT.html
Protocol #3 RT-PCR and PCR
Protocol #3. RT-PCR. Protocol #3. RT-PCR and PCR. Introduction. The RT-PCR kit contains polymerase and all necessary reagents to perform single tube RT and ...www.ucc.ie/ucc/depts/physio/MolPhysiol/MolPhys/Protocols/pdfs/003.pdf
RT-PCR Protocol ain this protocol is thermostable, reverse transcription time is cut in half. The speci-. ficity and sensitivity associated with RT-PCR is also heightened as ...www.mbpinc.com/html/pdf/products/pcr/RT-PCR%20Protocol%20a.pdf
RT PCRRT PCR PROTOCOL: BD BIOSCIENCES. . This protocol is adapted from a BDBiosciences protocol by the Gene Expression Lab. This protocol is for use with BD ...www.ncifcrf.gov/atp/LMT/RT_PCR_Protocol.pdf
Relative Quantitative RT-PCR Protocol.
Relative Quantitative RT-PCR Protocol. Wild-type and experimental plants should be planted and grown in parallel. Wild-type and experimental plants’ RNA ...www.chromatin-consortium.org/docs/rt-pcr_protocol.pdf
Protocol: a highly sensitive RT-PCR Method for Detection and Quantification of mRNAs.
Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and ...www.plantmethods.com/content/3/1/12
QIAGEN OneStep RT-PCR Handbook
This protocol serves as a guideline for one-step RT-PCR. ...... The QIAGEN OneStep RT-PCR Protocol is optimized for amplification of products of up ...www1.qiagen.com/HB/OneStepRTPCR
RT-PCR: Two-Step Protocol Two-step Protocol
RT-PCR: Two-Step Protocol. We will provide both one-step and two-step protocols ... In the one-step protocol, the components of RT and PCR are mixed in a ...ocw.mit.edu/NR/rdonlyres/Biology/7-16Spring-2005/85278F14-478B-469E-8D2E
Atlas Arrays RT-PCR User Manual
User Manual provides an RT-PCR protocol and related information for use .... RT-PCR Protocol continued. 11. The cDNA is now ready for immediate use or ...www.clontech.com/images/pt/PT3270-1.pdf
cDNA Synthesis for RT-PCR Protocol Reagents Procedure
cDNA Synthesis for RT-PCR Protocol. Section of Cancer Genomics, Genetics Branch, NCI. National Institutes of Health. Reagents. 5X First Strand Buffer ...www.riedlab.nci.nih.gov/publications/cDNA%20Synthesis%20for%20RT-PCR.pdf
Ambion: Automation of One-Step RT-PCR Using Cells-to-cDNA II
This automated protocol includes cell lysis, DNase I treatment, and set-up for one-step RT-PCR, that can then be used in gel-based or quantitative ...www.ambion.com/techlib/posters/c2cdna_automation_0204.html
Protocol for Preparation of DNA-free RNA Prior to RT-PCR
Protocol for Preparation of DNA-free RNA Prior to RT-PCR (with DNase I, RNase-free). Add to an RNase-free tube:. RNA, 1 µg. 10X reaction buffer with MgCl2 ...www.fermentas.com/techinfo/modifyingenzymes/protocols/p_prepdnafreerna.htm
Protocol Using QIAGEN OneStep RT-PCR Kit
This protocol serves as a guideline for one-step RT-PCR. .... QIAGEN OneStep RT-PCR. Protocol. Table 2. Thermal cycler conditions. Additional comments ...www.uark.edu/ua/henrylab/Links/biochemgen/QIAGEN%20RT-PCR%20protocol.pdf
rt pcr animation
RT-PCR stands for Reverse Transcription-Polymerase Chain Reaction. ... with primers and Taq Polymerase, and the standard PCR protocol is followed. ...www.bio.davidson.edu/people/kabernd/seminar/2002/method/lowry/RTPCR.htm
PCRboost Quick Reference Protocol
When using PCRboost, there is no change to your PCR or. RT-PCR protocol. Simply substitute PCRboost for water. The following are examples of PCR and RT-PCR ...www.biomatrica.com/media/PCRBoostProtocols.pdf
Two-Step RT-PCR Kit
PROTOCOL A:TWO-STEP RT-PCR. This standard protocol applies to a single ..... RT-PCR is carried out according to the standard protocol with 100 ng total RNA ...www.usbweb.com/proto/78355a.pdf
DNA Contamination in RT-PCR (Technical Bulletin #176)
Ways to get rid of DNA contamination in RNA samples--a frequent cause of concern among investigators performing quantitative RT-PCR.www.ambion.com/techlib/tb/tb_176.html
Reverse Transcriptase-PCR
Reverse Transcriptase-PCR Lazo Lab/Jim Hutchins
http://wheat.pw.usda.gov/~lazo/methods/lazo/revtrpcr.html
RT-PCR of Undifferentiated Cells [Stem Cell Information]
Skip to content. Click to visit the National Institutes of Health Web site · Stem Cell Information. Stem Cell Information. The National Institutes of Health ...stemcells.nih.gov/research/NIHresearch/scunit/RTPCR.asp
Web Source of PCR (3)
PCR Web Source
PCR Amplification of DNA
Department of Biological Sciences, University of Maryland, Baltimore County
Standard PCR Protocol
Molecular Biology Techniques Manual
Polymerase Chain Reaction
Dr. Chen, University College London (UK)
Protocol for PCR
MBI Fermentas
Polymerase Chain Reaction
Dr. H Donis-Keller, Washington University in St. Louis
Purification of Synthetic Oligonucleotides by Ion Exchange Chromatography
Dr. H Donis-Keller, Washington University in St. Louis
PCR Manual, 2nd Edition (complete)
Roche Molecular Biochemicals
PCR Product Isolation using SPRI
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology
PCR
Dr. DDL Bowtell, University of Melbourne (Australia)
Double-stranded and Single-stranded PCR
Dept. of Organismic and Evolutionary Biology, Harvard University
Calculating Concentrations for PCR
Molecular Biology Techniques Manual
Introduction to Polymerase Chain Reaction (PCR)
Dr. M Blader, Florida State University
Introduction to Polymerase Chain Reaction (PCR) Part II
Dr. M Blader, Florida State University
Guidelines for PCR
Qiagen
PCR Technology
Accessexcellence.com
Add
PCR Technology
LaboratoryExperiments.com
POLYMERASE CHAIN REACTION
Dr. DDL Bowtell, University of Melbourne (Australia)
PCR amplification of subclone inserts
Genome Sequencing Center, Washington University in St. Louis
Selective amplification of specific sequences of DNA from total genomic DNA using the polymerase chain reaction (PCR)
Dr. R Cruickshank, University of Glasgow (UK)
Designing PCR programs
Dr. Tavis, Yale University
PCR Trouble Shooting
Alkami Biosystems
RAPD PCR Colony Miniprep
Dr. GR Lazo, United States Department of Agriculture
RAPD PCR Reaction Mixes and Conditions
Dr. GR Lazo, United States Department of Agriculture
SINGLE-FLY DNA PREPS FOR PCR
Dr. GR Lazo, United States Department of Agriculture
PCR amplification of subclone inserts
Bioexchange.com
Purification of PCR fragments for cloning
Bioexchange.com
Degenerate PCR
Bioexchange.com
Polymerase Chain Reaction (PCR)
Bioexchange.com
AGAROSE GEL ELECTROPHORESIS OF PCR PRODUCTS
Current Protocols in Cell Biology, John Wiley & Sons, Inc.
PCR AMPLIFICATION OF RNA UNDER OPTIMAL CONDITIONS
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
ENZYMATIC AMPLIFICATION OF DNA BY PCR: STANDARD PROCEDURES AND OPTIMIZATION
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
PCR amplification of RNA: INTRODUCING cDNA DIRECTLY INTO THE AMPLIFICATION STEP
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
PCR amplification of RNA: AVOIDING LENGTHY COPRECIPITATION AND ANNEALING STEPS IN PCR OF RNA
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
General Notes on Primer Design in PCR
Brinkmann Instruments, Inc
Using Gradient PCR to determine the optimum annealing temperature
Brinkmann Instruments, Inc
Polymerase Chain Reaction (PCR)
Dr. P Bowyer, BBSRC, Long Ashton Research Station (UK)
BEST PCR conditions for amplifying DNA from plasmids
Dr. DE Koshland, Carnegie Institution of Washington
RAPID PREPARATION OF CRUDE RNA
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
PCR Amplification of DNA
Department of Biological Sciences, University of Maryland, Baltimore County
Standard PCR Protocol
Molecular Biology Techniques Manual
Polymerase Chain Reaction
Dr. Chen, University College London (UK)
Protocol for PCR
MBI Fermentas
Polymerase Chain Reaction
Dr. H Donis-Keller, Washington University in St. Louis
Purification of Synthetic Oligonucleotides by Ion Exchange Chromatography
Dr. H Donis-Keller, Washington University in St. Louis
PCR Manual, 2nd Edition (complete)
Roche Molecular Biochemicals
PCR Product Isolation using SPRI
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology
PCR
Dr. DDL Bowtell, University of Melbourne (Australia)
Double-stranded and Single-stranded PCR
Dept. of Organismic and Evolutionary Biology, Harvard University
Calculating Concentrations for PCR
Molecular Biology Techniques Manual
Introduction to Polymerase Chain Reaction (PCR)
Dr. M Blader, Florida State University
Introduction to Polymerase Chain Reaction (PCR) Part II
Dr. M Blader, Florida State University
Guidelines for PCR
Qiagen
PCR Technology
Accessexcellence.com
Add
PCR Technology
LaboratoryExperiments.com
POLYMERASE CHAIN REACTION
Dr. DDL Bowtell, University of Melbourne (Australia)
PCR amplification of subclone inserts
Genome Sequencing Center, Washington University in St. Louis
Selective amplification of specific sequences of DNA from total genomic DNA using the polymerase chain reaction (PCR)
Dr. R Cruickshank, University of Glasgow (UK)
Designing PCR programs
Dr. Tavis, Yale University
PCR Trouble Shooting
Alkami Biosystems
RAPD PCR Colony Miniprep
Dr. GR Lazo, United States Department of Agriculture
RAPD PCR Reaction Mixes and Conditions
Dr. GR Lazo, United States Department of Agriculture
SINGLE-FLY DNA PREPS FOR PCR
Dr. GR Lazo, United States Department of Agriculture
PCR amplification of subclone inserts
Bioexchange.com
Purification of PCR fragments for cloning
Bioexchange.com
Degenerate PCR
Bioexchange.com
Polymerase Chain Reaction (PCR)
Bioexchange.com
AGAROSE GEL ELECTROPHORESIS OF PCR PRODUCTS
Current Protocols in Cell Biology, John Wiley & Sons, Inc.
PCR AMPLIFICATION OF RNA UNDER OPTIMAL CONDITIONS
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
ENZYMATIC AMPLIFICATION OF DNA BY PCR: STANDARD PROCEDURES AND OPTIMIZATION
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
PCR amplification of RNA: INTRODUCING cDNA DIRECTLY INTO THE AMPLIFICATION STEP
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
PCR amplification of RNA: AVOIDING LENGTHY COPRECIPITATION AND ANNEALING STEPS IN PCR OF RNA
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
General Notes on Primer Design in PCR
Brinkmann Instruments, Inc
Using Gradient PCR to determine the optimum annealing temperature
Brinkmann Instruments, Inc
Polymerase Chain Reaction (PCR)
Dr. P Bowyer, BBSRC, Long Ashton Research Station (UK)
BEST PCR conditions for amplifying DNA from plasmids
Dr. DE Koshland, Carnegie Institution of Washington
RAPID PREPARATION OF CRUDE RNA
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
Special PCR Protocols
(from biogate)
384-well PCR
Adjuvants in PCR reactions
Alpha-satellite DNA by PCR Preparation
Amplification of Genomic DNA using Alu PCR
Calculating Concentrations for PCR
Choice of Polymerases for PCR
Colony PCR
Core Sample PCR
Degenerate PCR
Degenerate PCR Primer Design
Degenerate PCR, a short guide
Designing PCR programs
Direct PCR from Whole Yeast Cells: Zymolyase Method
Disruption by Fusion PCR
Home-made Taq Polymerase Purification
Incorporation of Digoxigenin-dUTP into Plasmid Inserts Using PCR
Inverse PCR
Inverse PCR
Inverse PCR & Cycle Sequencing of P Element Insertions for STS Generation
Inverse PCR for PAC-end sequencing
Long PCR Reagents and Guidelines
Long-PCR Reagents and Guidelines
Methylated CpG Island Amplification
Methylation-Specific PCR
Multiplex PCR: Critical Parameters and Step-by-Step Protocol
PCR Additives
PCR Amplification of DNA
PCR Amplification of Inserts from Bacterial Cultures
PCR and multiplex PCR guide
PCR and multiplex PCR Troubleshooting
PCR of blood, hair or small tissue samples
PCR Primer Design
PCR Primer Design and Reaction Optimization
PCR protocol
PCR Technology
PCR to Amplify rRNA Gene Fragment
PEG Precipitation of PCR products
Polymerase Chain Reaction
Primary Amplification of Genomic DNA using DOP - PCR
Primer Design
primer design for PCR cloning
Purification of PCR products with Sephadex
Quantitative RT-PCR and Other PCR Procedures
RT In Situ PCR
Singel Nucleotide Primer Extension (SNuPE)
Single Primer ("Semi-Random") PCR
Single Tube Confirmation PCR Protocol
Site-directed Mutagenesis using PCR
SOEing PCR for mutagenesis
Standard PCR Protocol
Tail DNA for PCR (No Organic Solvents)
The In Situ PCR: Amplification and Detection in a Cellular Context
384-well PCR
Adjuvants in PCR reactions
Alpha-satellite DNA by PCR Preparation
Amplification of Genomic DNA using Alu PCR
Calculating Concentrations for PCR
Choice of Polymerases for PCR
Colony PCR
Core Sample PCR
Degenerate PCR
Degenerate PCR Primer Design
Degenerate PCR, a short guide
Designing PCR programs
Direct PCR from Whole Yeast Cells: Zymolyase Method
Disruption by Fusion PCR
Home-made Taq Polymerase Purification
Incorporation of Digoxigenin-dUTP into Plasmid Inserts Using PCR
Inverse PCR
Inverse PCR
Inverse PCR & Cycle Sequencing of P Element Insertions for STS Generation
Inverse PCR for PAC-end sequencing
Long PCR Reagents and Guidelines
Long-PCR Reagents and Guidelines
Methylated CpG Island Amplification
Methylation-Specific PCR
Multiplex PCR: Critical Parameters and Step-by-Step Protocol
PCR Additives
PCR Amplification of DNA
PCR Amplification of Inserts from Bacterial Cultures
PCR and multiplex PCR guide
PCR and multiplex PCR Troubleshooting
PCR of blood, hair or small tissue samples
PCR Primer Design
PCR Primer Design and Reaction Optimization
PCR protocol
PCR Technology
PCR to Amplify rRNA Gene Fragment
PEG Precipitation of PCR products
Polymerase Chain Reaction
Primary Amplification of Genomic DNA using DOP - PCR
Primer Design
primer design for PCR cloning
Purification of PCR products with Sephadex
Quantitative RT-PCR and Other PCR Procedures
RT In Situ PCR
Singel Nucleotide Primer Extension (SNuPE)
Single Primer ("Semi-Random") PCR
Single Tube Confirmation PCR Protocol
Site-directed Mutagenesis using PCR
SOEing PCR for mutagenesis
Standard PCR Protocol
Tail DNA for PCR (No Organic Solvents)
The In Situ PCR: Amplification and Detection in a Cellular Context
Variants of PCR (4)
PCR animated - Animation illustrating the principle of PCR, from the University of Ghent, Belgium.
PCR Gateway - A directory of PCR techniques, PCR protocols, PCR troubleshooting, PCR websites and online resources from the publisher Horizon Press.
PCR Guru - A downloadable textbook on PCR setup and optimization, not free.
PCR method protocols - Protocols for PCR posted by the Helen Donis-Keller Laboratory.
PCR primer design and reaction optimisation - Article by Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town in: Molecular Biology Techniques Manual, on the web site of the University of Cape Town.
PCR Project - Presentations from the University of California at Berkley on PCR, both current research reports and reviews.
PCR Protocol - Detailed PCR protocol from the web site of the Department of Biology, University of Michigan, USA.
PCR Protocols - Protocols and technical hints, particularly for reverse transcription PCR, somewhat outdated, compiled by Dr Jack Vanden Heuvel, Department of Veterinary Science and Molecular Toxicology Program, Penn State University
PCR Technology - An introduction by Connie Veilleux from the US National Health Museum website.
PCR Troubleshooting - Limited to conventional straight forward PCR. Page designed and maintained by Octavian Henegariu on the web site of the Yale - New Haven Medical Center.
PCR-ELISA and Related - P Zhang, CJ Gebhart, D Burden, GE Duhamel: A low technology alternative to real time PCR, technical article on the site of BT&C, Inc Bridgewater, NJ, USA.
Polymerase Chain Reaction - Popular survey article by Mark V. Bloom, DNA Learning Center, Cold Spring Harbor Laboratory, from the web site of the US National Health Museum.
Polymerase Chain Reaction (PCR) - A graphic description of the principle of PCR from the US National Health Museum web site.
Principle of PCR - Applications in work on aging of Caenorhabditis elegans and phylogeny of nematodes, by Andy Vierstraete, Department of Biology, University of Ghent, Belgium.
Protocols Online: PCR Protocols - Extensive collection of PCR protocols and methods from Protocol On Line.
Quantitative PCR Protocol - From the Jackson Laboratory, University of Maine, USA.
Random Arbitrarily Primed PCR - Protocol for RAP-PCR to study prokaryotic gene expression, from the Enterococcus Research Site, University of Oklahoma, USA.
RAPD analysis with P. infestans - Protocol for Randomly Amplified Polymorphic DNA, by H Judelson, University of California, Riverside, USA.
RAPD PCR - RAPD stands for Random Amplification of Polymorphic DNA, where the target sequence(s) (to be amplified) is unknown.Brief description, from Rutgers University, USA.
RAPD's - Brief description of Random Amplified Polymorphic DNA, from Oklahoma State University, USA.
Rational primer design greatly improves differential display-PCR (DD-PCR) - Article: D Graf, AG Fisher, M Merkenschlager: Nucl. Acids Res. 25:11 2239-2240.
Reference in PCR - Technical aspects of quantitative real-time PCR and RT-PCR. Instruments, kits, dyes, chemistries, and services presented by their manufacturers.
Rep-PCR Genomic Fingerprinting - Bacteria are characterized by Rep-PCR fingerprinting using primers corresponding to naturally occurring repetitive sequences in the interspersed regions.
RFLP Definition - RFLP = Restriction Fragment Length Polymorphism, from FDA
Roe Laboratory Protocols - Molecular biological protocols, mostly PCR related used by Bruce A. Roe at the Dept. of Chemistry and Biochemistry, OU, Norman, OK.
Single tube confirmation PCR protocol - For characterization colonies of transformed clones of Saccharaomyces, from the web site of the Stanford Genome Technology Center, Palo Alto, CA, USA.
Standard PCR protocols - From Molecular Biology Techniques Manual, from the web site of the University of Cape Town, South Africa.
Tavi's PCR protocols - A page describing the main parameters and trouble-shooting in PCR. The page is somewhat dated (updated 1997) but still useful.
T-DNA Generated Enhancer Traps in Arabidopsis - Application of inverse PCR, partial genomic libraries and TAIL-PCR in cloning flanking, at the Department of Biological Sciences, Dartmouth College, Hanover, NH.
Technical manual of PCR - Intended for specialists planning PCR procedures in their laboratories. From the University of Cape Town updated 2001.
The "Poison Primer" Technique for Enhancing Detection of Small Deletions in Mutant Libraries - This method works because the poison allows the formation of deletion products but titers out full-sized products. From the Biotechnology Laboratory, University of British Columbia.
The web guide of PCR - List of links and forum on the subject and related methodology. Set up and maintained by SJ Krivokapich, National University of Misiones, Argentina.
Thermostable DNA Polymerases - Discussion of their origin and briefly their properties. From the web site of Colorado State University.
Wayward PCR primers - Article by PN Hengen from TIBS 1995 on the loss of activity of PCR primers with time.
What the Heck is PCR? - Popular description of the PCR technique by John C Brown, University of Kansas 1995.
Which DNA Marker for Which Purpose? - Compendia of the Research Project "Development, optimisation and validation of molecular tools for assessment of biodiversity in forest trees", European Union DGXII Biotechnology FW IV Research Programme. From the web site of the University Library, Göttingen.
PCR Gateway - A directory of PCR techniques, PCR protocols, PCR troubleshooting, PCR websites and online resources from the publisher Horizon Press.
PCR Guru - A downloadable textbook on PCR setup and optimization, not free.
PCR method protocols - Protocols for PCR posted by the Helen Donis-Keller Laboratory.
PCR primer design and reaction optimisation - Article by Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town in: Molecular Biology Techniques Manual, on the web site of the University of Cape Town.
PCR Project - Presentations from the University of California at Berkley on PCR, both current research reports and reviews.
PCR Protocol - Detailed PCR protocol from the web site of the Department of Biology, University of Michigan, USA.
PCR Protocols - Protocols and technical hints, particularly for reverse transcription PCR, somewhat outdated, compiled by Dr Jack Vanden Heuvel, Department of Veterinary Science and Molecular Toxicology Program, Penn State University
PCR Technology - An introduction by Connie Veilleux from the US National Health Museum website.
PCR Troubleshooting - Limited to conventional straight forward PCR. Page designed and maintained by Octavian Henegariu on the web site of the Yale - New Haven Medical Center.
PCR-ELISA and Related - P Zhang, CJ Gebhart, D Burden, GE Duhamel: A low technology alternative to real time PCR, technical article on the site of BT&C, Inc Bridgewater, NJ, USA.
Polymerase Chain Reaction - Popular survey article by Mark V. Bloom, DNA Learning Center, Cold Spring Harbor Laboratory, from the web site of the US National Health Museum.
Polymerase Chain Reaction (PCR) - A graphic description of the principle of PCR from the US National Health Museum web site.
Principle of PCR - Applications in work on aging of Caenorhabditis elegans and phylogeny of nematodes, by Andy Vierstraete, Department of Biology, University of Ghent, Belgium.
Protocols Online: PCR Protocols - Extensive collection of PCR protocols and methods from Protocol On Line.
Quantitative PCR Protocol - From the Jackson Laboratory, University of Maine, USA.
Random Arbitrarily Primed PCR - Protocol for RAP-PCR to study prokaryotic gene expression, from the Enterococcus Research Site, University of Oklahoma, USA.
RAPD analysis with P. infestans - Protocol for Randomly Amplified Polymorphic DNA, by H Judelson, University of California, Riverside, USA.
RAPD PCR - RAPD stands for Random Amplification of Polymorphic DNA, where the target sequence(s) (to be amplified) is unknown.Brief description, from Rutgers University, USA.
RAPD's - Brief description of Random Amplified Polymorphic DNA, from Oklahoma State University, USA.
Rational primer design greatly improves differential display-PCR (DD-PCR) - Article: D Graf, AG Fisher, M Merkenschlager: Nucl. Acids Res. 25:11 2239-2240.
Reference in PCR - Technical aspects of quantitative real-time PCR and RT-PCR. Instruments, kits, dyes, chemistries, and services presented by their manufacturers.
Rep-PCR Genomic Fingerprinting - Bacteria are characterized by Rep-PCR fingerprinting using primers corresponding to naturally occurring repetitive sequences in the interspersed regions.
RFLP Definition - RFLP = Restriction Fragment Length Polymorphism, from FDA
Roe Laboratory Protocols - Molecular biological protocols, mostly PCR related used by Bruce A. Roe at the Dept. of Chemistry and Biochemistry, OU, Norman, OK.
Single tube confirmation PCR protocol - For characterization colonies of transformed clones of Saccharaomyces, from the web site of the Stanford Genome Technology Center, Palo Alto, CA, USA.
Standard PCR protocols - From Molecular Biology Techniques Manual, from the web site of the University of Cape Town, South Africa.
Tavi's PCR protocols - A page describing the main parameters and trouble-shooting in PCR. The page is somewhat dated (updated 1997) but still useful.
T-DNA Generated Enhancer Traps in Arabidopsis - Application of inverse PCR, partial genomic libraries and TAIL-PCR in cloning flanking, at the Department of Biological Sciences, Dartmouth College, Hanover, NH.
Technical manual of PCR - Intended for specialists planning PCR procedures in their laboratories. From the University of Cape Town updated 2001.
The "Poison Primer" Technique for Enhancing Detection of Small Deletions in Mutant Libraries - This method works because the poison allows the formation of deletion products but titers out full-sized products. From the Biotechnology Laboratory, University of British Columbia.
The web guide of PCR - List of links and forum on the subject and related methodology. Set up and maintained by SJ Krivokapich, National University of Misiones, Argentina.
Thermostable DNA Polymerases - Discussion of their origin and briefly their properties. From the web site of Colorado State University.
Wayward PCR primers - Article by PN Hengen from TIBS 1995 on the loss of activity of PCR primers with time.
What the Heck is PCR? - Popular description of the PCR technique by John C Brown, University of Kansas 1995.
Which DNA Marker for Which Purpose? - Compendia of the Research Project "Development, optimisation and validation of molecular tools for assessment of biodiversity in forest trees", European Union DGXII Biotechnology FW IV Research Programme. From the web site of the University Library, Göttingen.
Variants of PCR (3)
A Rapid DNA Minipreparation Method Suitable for AFLP and Other PCR Applications (PDF document) - Preparation of DNA from plant tissues suitable for PCR methods including AFLP, article by DH CHEN and PC RONALD Department of Plant Pathology, University of California, Davis.
Adjuvants in PCR Reactions - Brief discussion of additives to improve amplification efficiency and specificity of PCR, by Octavian Henegariu, Yale-New Haven Medical Center.
Alu-PCR Hybridization - A report and protocol, from Fondation Jean Dausset.
Anchor Probes for Comparative Mapping of Grass Species - Article in which probes from different libraries were used to hybridize seven cereals at the Department of Plant Breeding and Biometry, Cornell University, NY.
Attotron Biosensor Corporation - R & D company for development of biosensors and related products for the research and educational markets.
BioRad, Amplification, PCR - Division of BioRad Laboratories that manufactures and sells instruments for PCR, in Hercules, California, USA.
Biosource molecular methods booklet - 56 page booklet on oligonucleotides, PCR and RT-PCR, from Biosource (company).
C. elegans Gene Knockout Project - Protocols including standard nested PCR, from the Biotechnology Laboratory, University of British Columbia.
Degenerate PCR - An introduction with principle, applications and description of the technique. From the web site of Kebangsaan University, Malaysia.
Degenerate PCR - The identification of novel members of gene families by PCR using degenerate primers is described and protocols given. Article by Michael Koelle 1996 on the web site of Dartmouth College.
Design Oligonunucleotide Primers - Interface for scanning DNA sequences for restriction sites, without changing encoded protein created. Includes FAQ, table of enzymes and links to tools, maintained at the University of Waterloo, ON, Canada.
Detection of Asymmetric PCR Products in Homogeneous Solution by Fluorescence Correlation Spectroscopy - In asymmetric PCR, the low concentration primer is quantitatively incorporated into double stranded DNA after an appropriate number of cycles. If this primer is fluorescent labelled, the dsDNA can be quantitated from the fluorescence.
Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences - RFLP = Restriction Fragment Length Polymorphism. By Alexander Binder 1997.
Detection of Single Nucleotide Mutations in Wheat Using Single Strand Conformation Polymorphism Gels (PDF file) - P Martins-Lopez, H Zhang, R Koebner, Plant Mol. Biol. Reporter 19(2001): 159-162. From National Research Coouncil Canada.
DNALC: PCR Animation - An animation explaining how the Polymerase Chain Reaction (PCR) works, from the Dolan DNA learning center, Cold Spring Harbor Laboratory, USA.
Dolan DNA Learning Centers Gene Almanac - Educational site on topics in genetics and gene expression from Cold Spring Harbor Laboratory, USA.
Effect of PCR Buffer on multiplex PCR (PDF) - Multiplex PCR employs different primer pairs in the same amplification reaction. This requires extensive optimization of annealing conditions. From Quiagen (company)
Fidelity of DNA Polymerases for PCR - Article by PN Hengen from TIBS 1995
FISH Guide and Troubleshooting - Links to pages describing influential parameters, with guides on PCR, RT-PCR and multiplex PCR reactions, Taq, FISH, CM-FISH, TM-FISH, microarrays, CCK, slide prep and labeling, maintained by Octavian Henegariu from Yale University, New Haven, CT.
GeneOhm Sciences - Tests on gruup B Streptococcus and methicillin resistant Staphylococcus aureus by PCR / DNA sequencing.
GenHunter - Manufacturer of material for differential display PCR in Nashville, Tenn USA.
HiFi DNA - HiFi DNA is a company selling a DNA polymerase for PCR at low temperature giving accurate replication of certain sequences where Taq fails.
Ingenetix GmbH - Develops technology and products for DNA and mRNA research. Also provide DNA testing for the determination of parentage/paternity and custom DNA sequencing, oligonucleotide synthesis, genotyping services, pharmacogenetics and quantitative PCR, in Vienna, Austria.
Inverse PCR and Cycle Sequencing of P Element Insertions for STS Generation - Step by step protocol, by EJ Rehm, Berkeley Drosophila Genome Project, USA.
Inverse PCR for PAC-end sequencing - To generate PCR fragments that contain the ends of PAC inserts that can be sequenced. Protocol by B Barbazuk, Washington University Zebrafish Genome Resources Project, USA.
Inverse PCR For use with Snyder mTn-lacZ/LEU2 Based Mutagenesis - Protocol by M McMurray, Fred Hutchinson Cancer Research Center, Seattle, Wa. USA.
Inverse PCR protocol - step by step protocol, from the web site of the Department of Biology, University of Michigan, USA.
Kary B. Mullis - Autobiography - The originator of PCR, from the Nobel e-museum web site.
Kary Mullis - Inventor Profile of Kary Mullis, the originator of PCR, from the National Inventors Hall of Fame web site.
Long PCR Protocol - Protocol and guidelines for choice of conditions for PCR of long sequences (10 kb or larger). From Genetics Dept., Harvard Medical School, Boston, MA, USA
Nematode ITS1 Size Variation - Examples of Restriction Fragment Length Polymorphism (RFLP)electrophoresis slabs for different nematodes, from University of Nebrasca.
Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler - Basic discussion of the optimization of PCR, from the web site of Brinkmann Instruments.
Optimizing Multiplex and LA-PCR with Betaine - LA-PCR = "long and accurate PCR". Article by PN Hengen in TIBS June 1997.
Optimizing PCR Protocols - Brief guidelines. From the Jackson Laboratory, University of Maine, USA.
PCR Amplification of cDNA Segments by 2 Stage Nested PCR - Protocol from the method database of NIH, USA.
PCR and multiplex PCR guide - Discussions of the parameters influencing the PCR reaction and some PCR and multiplex PCR applications, by Octavian Henegariu on the web site of the Yale - New Haven Medical center.
Adjuvants in PCR Reactions - Brief discussion of additives to improve amplification efficiency and specificity of PCR, by Octavian Henegariu, Yale-New Haven Medical Center.
Alu-PCR Hybridization - A report and protocol, from Fondation Jean Dausset.
Anchor Probes for Comparative Mapping of Grass Species - Article in which probes from different libraries were used to hybridize seven cereals at the Department of Plant Breeding and Biometry, Cornell University, NY.
Attotron Biosensor Corporation - R & D company for development of biosensors and related products for the research and educational markets.
BioRad, Amplification, PCR - Division of BioRad Laboratories that manufactures and sells instruments for PCR, in Hercules, California, USA.
Biosource molecular methods booklet - 56 page booklet on oligonucleotides, PCR and RT-PCR, from Biosource (company).
C. elegans Gene Knockout Project - Protocols including standard nested PCR, from the Biotechnology Laboratory, University of British Columbia.
Degenerate PCR - An introduction with principle, applications and description of the technique. From the web site of Kebangsaan University, Malaysia.
Degenerate PCR - The identification of novel members of gene families by PCR using degenerate primers is described and protocols given. Article by Michael Koelle 1996 on the web site of Dartmouth College.
Design Oligonunucleotide Primers - Interface for scanning DNA sequences for restriction sites, without changing encoded protein created. Includes FAQ, table of enzymes and links to tools, maintained at the University of Waterloo, ON, Canada.
Detection of Asymmetric PCR Products in Homogeneous Solution by Fluorescence Correlation Spectroscopy - In asymmetric PCR, the low concentration primer is quantitatively incorporated into double stranded DNA after an appropriate number of cycles. If this primer is fluorescent labelled, the dsDNA can be quantitated from the fluorescence.
Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences - RFLP = Restriction Fragment Length Polymorphism. By Alexander Binder 1997.
Detection of Single Nucleotide Mutations in Wheat Using Single Strand Conformation Polymorphism Gels (PDF file) - P Martins-Lopez, H Zhang, R Koebner, Plant Mol. Biol. Reporter 19(2001): 159-162. From National Research Coouncil Canada.
DNALC: PCR Animation - An animation explaining how the Polymerase Chain Reaction (PCR) works, from the Dolan DNA learning center, Cold Spring Harbor Laboratory, USA.
Dolan DNA Learning Centers Gene Almanac - Educational site on topics in genetics and gene expression from Cold Spring Harbor Laboratory, USA.
Effect of PCR Buffer on multiplex PCR (PDF) - Multiplex PCR employs different primer pairs in the same amplification reaction. This requires extensive optimization of annealing conditions. From Quiagen (company)
Fidelity of DNA Polymerases for PCR - Article by PN Hengen from TIBS 1995
FISH Guide and Troubleshooting - Links to pages describing influential parameters, with guides on PCR, RT-PCR and multiplex PCR reactions, Taq, FISH, CM-FISH, TM-FISH, microarrays, CCK, slide prep and labeling, maintained by Octavian Henegariu from Yale University, New Haven, CT.
GeneOhm Sciences - Tests on gruup B Streptococcus and methicillin resistant Staphylococcus aureus by PCR / DNA sequencing.
GenHunter - Manufacturer of material for differential display PCR in Nashville, Tenn USA.
HiFi DNA - HiFi DNA is a company selling a DNA polymerase for PCR at low temperature giving accurate replication of certain sequences where Taq fails.
Ingenetix GmbH - Develops technology and products for DNA and mRNA research. Also provide DNA testing for the determination of parentage/paternity and custom DNA sequencing, oligonucleotide synthesis, genotyping services, pharmacogenetics and quantitative PCR, in Vienna, Austria.
Inverse PCR and Cycle Sequencing of P Element Insertions for STS Generation - Step by step protocol, by EJ Rehm, Berkeley Drosophila Genome Project, USA.
Inverse PCR for PAC-end sequencing - To generate PCR fragments that contain the ends of PAC inserts that can be sequenced. Protocol by B Barbazuk, Washington University Zebrafish Genome Resources Project, USA.
Inverse PCR For use with Snyder mTn-lacZ/LEU2 Based Mutagenesis - Protocol by M McMurray, Fred Hutchinson Cancer Research Center, Seattle, Wa. USA.
Inverse PCR protocol - step by step protocol, from the web site of the Department of Biology, University of Michigan, USA.
Kary B. Mullis - Autobiography - The originator of PCR, from the Nobel e-museum web site.
Kary Mullis - Inventor Profile of Kary Mullis, the originator of PCR, from the National Inventors Hall of Fame web site.
Long PCR Protocol - Protocol and guidelines for choice of conditions for PCR of long sequences (10 kb or larger). From Genetics Dept., Harvard Medical School, Boston, MA, USA
Nematode ITS1 Size Variation - Examples of Restriction Fragment Length Polymorphism (RFLP)electrophoresis slabs for different nematodes, from University of Nebrasca.
Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler - Basic discussion of the optimization of PCR, from the web site of Brinkmann Instruments.
Optimizing Multiplex and LA-PCR with Betaine - LA-PCR = "long and accurate PCR". Article by PN Hengen in TIBS June 1997.
Optimizing PCR Protocols - Brief guidelines. From the Jackson Laboratory, University of Maine, USA.
PCR Amplification of cDNA Segments by 2 Stage Nested PCR - Protocol from the method database of NIH, USA.
PCR and multiplex PCR guide - Discussions of the parameters influencing the PCR reaction and some PCR and multiplex PCR applications, by Octavian Henegariu on the web site of the Yale - New Haven Medical center.
Important Publications at Cell
1 . Molecular Interactions between Bacterial Symbionts and Their HostsColin Dale and Nancy A. MoranCell 126: 453-465.[Full Text] [PDF]
Symbiotic bacteria are important in animal hosts, but have been largely overlooked as they have proved difficult to culture in the laboratory. Approaches such as comparative genomics and real-time PCR have provided insights into the molecular mechanisms that underpin symbiont-host interactions. Studies on the heritable symbionts of insects have yielded valuable information about how bacteria infect host cells, avoid immune responses, and manipulate host physiology. Furthermore, some symbionts use many of the same mechanisms as pathogens to infect hosts and evade immune responses. Here we discuss what is currently known about the interactions between bacterial symbionts and their hosts.
2 . Loss of ARNT/HIF1β Mediates Altered Gene Expression and Pancreatic-Islet Dysfunction in Human Type 2 DiabetesJenny E. Gunton, Rohit N. Kulkarni, SunHee Yim, Terumasa Okada, Wayne J. Hawthorne, Yu-Hua Tseng, Russell S. Roberson, Camillo Ricordi, Philip J. O’Connell, Frank J. Gonzalez and C. Ronald KahnCell 122: 337-349.[Full Text] [PDF]
β cell dysfunction is a central component of the pathogenesis of type 2 diabetes. Using oligonucleotide microarrays and real-time PCR of pancreatic islets isolated from humans with type 2 diabetes versus normal glucose-tolerant controls, we identified multiple changes in expression of genes known to be important in β cell function, including major decreases in expression of HNF4α, insulin receptor, IRS2, Akt2, and several glucose-metabolic-pathway genes. There was also a 90% decrease in expression of the transcription factor ARNT. Reducing ARNT levels in Min6 cells with small interfering RNA (siRNA) resulted in markedly impaired glucose-stimulated insulin release and changes in gene expression similar to those in human type 2 islets. Likewise, β cell-specific ARNT knockout mice exhibited abnormal glucose tolerance, impaired insulin secretion, and changes in islet gene expression that mimicked those in human diabetic islets. Together, these data suggest an important role for decreased ARNT and altered gene expression in the impaired islet function of human type 2 diabetes.
3 . From Microbes to Prions: The Final Proof of the Prion HypothesisWen-Quan Zou and Pierluigi GambettiCell 121: 155-157.[Full Text] [PDF]
Much like the “microbe hypothesis” put forth over 150 years ago, the “prion hypothesis” can be definitely proven only if a prion disease is engendered in a natural host from an infectious prion produced in vitro. In this issue of Cell, Akman et al., 2002Abbott et al., 2000Castilla et al., 2005Blackburn, 1992 come very close to accomplishing this goal by producing a prion disease in a natural host from a prion entirely generated in vitro using a PCR-like amplification system.
4 . Microarray Identification of FMRP-Associated Brain mRNAs and Altered mRNA Translational Profiles in Fragile X SyndromeVictoria Brown, Peng Jin, Stephanie Ceman, Jennifer C. Darnell, William T. O'Donnell, Scott A. Tenenbaum, Xiaokui Jin, Yue Feng, Keith D. Wilkinson, Jack D. Keene, Robert B. Darnell and Stephen T. WarrenCell 107: 477-487.[Full Text] [PDF]
Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.
5 . RNAi as Random Degradative PCR: siRNA Primers Convert mRNA into dsRNAs that Are Degraded to Generate New siRNAsConcetta Lipardi, Qin Wei and Bruce M. PatersonCell 107: 297-307.[Full Text] [PDF]
In posttranscriptional gene silencing (PTGS), “quelling,” and RNA interference (RNAi), 21–25 nucleotide RNA fragments are produced from the initiating dsRNA. These short interfering RNAs (siRNAs) mediate RNAi by an unknown mechanism. Here, we show that GFP and Pp-Luc siRNAs, isolated from a protein complex in Drosophila embryo extract, target mRNA degradation in vitro. Most importantly, these siRNAs, as well as a synthetic 21-nucleotide duplex GFP siRNA, serve as primers to transform the target mRNA into dsRNA. The nascent dsRNA is degraded to eliminate the incorporated target mRNA while generating new siRNAs in a cycle of dsRNA synthesis and degradation. Evidence is presented that mRNA-dependent siRNA incorporation to form dsRNA is carried out by an RNA-dependent RNA polymerase activity (RdRP).
6 . Combinatorial Receptor Codes for OdorsBettina Malnic, Junzo Hirono, Takaaki Sato and Linda B Buck96: 713-723.[Full Text] [PDF]
The discriminatory capacity of the mammalian olfactory system is such that thousands of volatile chemicals are perceived as having distinct odors. Here we used a combination of calcium imaging and single-cell RT–PCR to identify odorant receptors (ORs) for odorants with related structures but varied odors. We found that one OR recognizes multiple odorants and that one odorant is recognized by multiple ORs, but that different odorants are recognized by different combinations of ORs. Thus, the olfactory system uses a combinatorial receptor coding scheme to encode odor identities. Our studies also indicate that slight alterations in an odorant, or a change in its concentration, can change its “code,” potentially explaining how such changes can alter perceived odor quality.
7 . Requirement for Specific Proteases in Cancer Cell Intravasation as Revealed by a Novel Semiquantitative PCR-Based AssayJ Kim, W Yu, K Kovalski and L Ossowski94: 353-362.[Full Text] [PDF]
Proteases are crucial for cancer metastasis, but due to lack of assays, their role in intravasation has not yet been tested. We have developed a human Alu sequence PCR-based assay to quantitate intravasated cells in an in vivo model. We demonstrated that metalloproteinases (MMPs), and most likely MMP-9, are required for intravasation by showing that marimastat, an inhibitor of MMPs, reduced intravasation by more than 90%, and that only tumor cell lines expressing MMP-9 intravasated. Cells with low surface urokinase plasminogen activator (uPA) and uPA receptor (uPAR) were also incapable of intravasation, despite the presence of high levels of MMP-9. We concluded that breaching of the vascular wall is a rate-limiting step for intravasation, and consequently for metastasis, and that cooperation between uPA/uPAR and MMP-9 is required to complete this step.
8 . Neandertal DNA Sequences and the Origin of Modern HumansMatthias Krings, Anne Stone, Ralf W Schmitz, Heike Krainitzki, Mark Stoneking and Svante Pääbo90: 19-30.[Full Text] [PDF]
DNA was extracted from the Neandertal-type specimen found in 1856 in western Germany. By sequencing clones from short overlapping PCR products, a hitherto unknown mitochondrial (mt) DNA sequence was determined. Multiple controls indicate that this sequence is endogenous to the fossil. Sequence comparisons with human mtDNA sequences, as well as phylogenetic analyses, show that the Neandertal sequence falls outside the variation of modern humans. Furthermore, the age of the common ancestor of the Neandertal and modern human mtDNAs is estimated to be four times greater than that of the common ancestor of human mtDNAs. This suggests that Neandertals went extinct without contributing mtDNA to modern humans.
9 . Altered Ca2+ Responses in Muscles with Combined Mitochondrial and Cytosolic Creatine Kinase DeficienciesKaren Steeghs, Ad Benders, Frank Oerlemans, Arnold de Haan, Arend Heerschap, Wim Ruitenbeek, Carolina Jost, Jan van Deursen, Benjamin Perryman, Dirk Pette, Marloes Brückwilder, Jolande Koudijs, Paul Jap, Jacques Veerkamp and Bé Wieringa89: 93-103.[Full Text] [PDF]
We have blocked creatine kinase (CK)-mediated phosphocreatine (PCr) ATP transphosphorylation in skeletal muscle by combining targeted mutations in the genes encoding mitochondrial and cytosolic CK in mice. Contrary to expectation, the PCr level was only marginally affected, but the compound was rendered metabolically inert. Mutant muscles in vivo showed significantly impaired tetanic force output, increased relaxation times, altered mitochondrial volume and location, and conspicuous tubular aggregates of sarcoplasmic reticulum membranes, as seen in myopathies with electrolyte disturbances. In depolarized myotubes cultured in vitro, CK absence influenced both the release and sequestration of Ca2+. Our data point to a direct link between the CK–PCr system and Ca2+-flux regulation during the excitation and relaxation phases of muscle contraction.
10 . Beyond PCR: Biotechnology as an Emerging CultureAdam Telerman and Robert B Amson86: 707-708.[Full Text] [PDF] 11 . The FHIT Gene at 3p14.2 Is Abnormal in Lung CancerGabriella Sozzi, Maria Luisa Veronese, Massimo Negrini, Raffaele Baffa, Maria Grazia Cotticelli, Hiroshi Inoue, Silvana Tornielli, Silvana Pilotti, Laura De Gregorio, Ugo Pastorino, Marco A Pierotti, Masataka Ohta, Kay Huebner and Carlo M Croce85: 17-26.[Full Text] [PDF]
To determine the role of the FHIT gene, which encompasses the fragile site at 3p14.2, we analyzed 59 tumors of the small cell and non-small cell type by reverse transcription of FHIT mRNA, followed by PCR amplification and sequencing of products. Allelic losses affecting the gene were evaluated by microsatellite polymorphism analysis and genomic alterations by hybridization using cDNA and genomic probes. Small cell lung tumors (80%) and non-small cell lung cancers (40%) showed abnormalities in RNA transcripts of FHIT, and 76% of the tumors exhibited loss of FHIT alleles. Abnormal lung tumor transcripts lack two or more exons of the FHIT gene. Small cell lung cancer tumors and cell lines were analyzed by Southern blotting and showed rearranged BamHI fragments. These data suggest a critical role of the FHIT gene in lung carcinogenesis.
12 . An in vitro assay for saccharomyces telomerase requires EST1Jing-Jer Lin and Virginia A. Zakian81: 1127-1135.[PDF]
Telomerase activity was demonstrated in cell-free extracts from S. cerevisiae through the use of a PCR-based assay. As expected, this activity was eliminated by RNase or phenol treatment of the extract and was dependent on dGTP and dTTP. Telomerase was not detected in extracts prepared from cells grown for ∼ 30 or more cell divisions in the absence of the EST1 product, Est1 p. TLC1 RNA, which determines the sequence of telomeric DNA in vivo, was present in normal amounts in est1Δ cells. Moreover, TLC1 RNA specifically precipitated with epitope-tagged Est1p. These data indicate that Estlp is either a subunit of yeast telomerase or an accessory protein associated with telomerase that is essential in vitro for its activity.
13 . A novel form of Epstein-Barr virus latency in normal B cells in vivoEmily M Miyashita, Bin Yang, Kitty M.C Lam, Dorothy H Crawford and David A Thorley-LawsonCell 80: 593-601.[PDF]
We have developed a PCR assay that can detect a single Epstein-Barr virus (EBV) genome in the presence of 106 uninfected cells. Using this assay, we demonstrate that EBV persists, in the peripheral blood of all seropositive individuals tested, in CD19+, CD23−, and CD80 (B7)− B cells. We further show that the virus in these cells is latent, but readily reactivated to produce infectious immortalizing virus; therefore, these cells represent a true site of latent persistence. EBV was not significantly detected in monocytes or T cells. The frequency of infected cells in nine healthy donors varied from 23 to 625 per 107 B cells, but was relatively stable for each individual over the course of 2 years. We conclude that the EBV-infected cells in vivo are B cells with a nonactivated phenotype. This represents a novel form of latency in normal B cells.
14 . The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophyNatalie Roy, Mani S Mahadevan, Michael McLean, Gary Shutter, Zahra Yaraghi, Reza Farahani, Stephen Baird, Anne Besner-Johnston, Charles Lefebvre, Xiaolin Kang, Maysoon Salih, Huguette Aubry, Katsuyuki Tamai, Xiaoping Guan, Panayiotis Ioannou, Thomas O Crawford, Pieter J de Jong, Linda Surh, Joh-E Ikeda, Robert G Korneluk and Alex MacKenzieCell 80: 167-178.[PDF]
The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR. analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.
15 . HIV and T cell expansion in splenic white pulps is accompanied by infiltration of HIV-specific cytotoxic T lymphocytesRémi Cheynier, Sven Henrichwark, Fabienne Hadida, Eric Pelletier, Eric Oksenhendler, Brigitte Autran and Simon Wain-HobsonCell 78: 373-387.[PDF]
Human immunodeficiency virus (HIV) replication and T cell proliferation were investigated in situ by a PCR-based analysis of individual microdissected splenic white pulps. Founder effects, revealed by an exquisite compartmentalization of HIV genotypes and T cells, indicated the recruitment of latently infected CD4+ T cells through highly localized antigen presentation rather than the infection of CD4+ T lymphoblasts by blood-borne virus or immune complexes. HIV-infected white pulps could be infiltrated by HIV-specific cytotoxic T lymphocytes, thereby implicating them in CD4+ T cell destruction in vivo. Together these data describe an iterative and deleterious mechanism of antigen-driven T cell recruitment and activation, as well as HIV replication and spread, with consequent destruction of the newly infected cells.
16 . Skeletal muscles of mice deficient in muscle creatine kinase lack burst activityJan van Deursen, Arend Heerschap, Frank Oerlemans, Wim Rultenbeek, Paul Jap, Henk ter Laak and Bé WieringaCell 74: 621-631.[PDF]
To understand the physiological role of the creatine kinase-phosphocreatine (CK-PCr) system in muscle bioenergetics, a null mutation of the muscle CK (M-CK) gene was introduced into the germline of mice. Mutant mice show no alterations in absolute muscle force, but lack the ability to perform burst activity. Their fast-twitch fibers have an increased intermyofibrillar mitochondrial volume and an increased glycogenolytic/glycolytic potential. PCr and ATP levels are normal in resting M-CK-deficient muscles, but rates of high energy phosphate exchange between PCr and ATP are at least 20-fold reduced. Strikingly, PCr levels decline normally during muscle exercise, suggesting that M-CK-mediated conversion is not the only route for PCr utilization in active muscle.
17 . Hotspots for unselected Ty1 transposition events on yeast chromosome III are near tRNA genes and LTR sequencesH. Ji, D.P. Moore, M.A. Blomberg, L.T. Braiterman, D.F. Voytas, G. Natsoulis and J.D. BoekeCell 73: 1007-1018.[PDF]
A collection of yeast strains bearing single marked Ty1 insertions on chromosome III was generated. Over 100 such insertions were physically mapped by pulsed-field gel electrophoresis. These insertions are very nonrandomly distributed. Thirty-two such insertions were cloned by the inverted PCR technique, and the flanking DNA sequences were determined. The sequenced insertions all fell within a few very limited regions of chromosome III. Most of these regions contained tRNA coding regions and/or LTRs of preexisting transposable elements. Open reading frames were disrupted at a far lower frequency than expected for random transposition. The results suggest that the Ty1 integration machinery can detect regions of the genome that may represent “safe havens” for insertion. These regions of the genome do not contain any special DNA sequences, nor do they behave as particularly good targets for Ty1 integration in vitro, suggesting that the targeted regions have special properties allowing specific recognition in vivo.
18 . Mapping the whole human genome by fingerprinting yeast artificial chromosomesChristine Bellanné-Chantelot, Bruno Lacroix, Pierre Ougen, Alain Billault, Sandrine Beaufils, Stéphane Bertrand, Isabelle Georges, Fabrice Glibert, Isabelle Gros, Georges Lucotte, Laurent Susini, Jean-Jacques Codani, Philippe Gesnouin, Stuart Pook, Guy Vaysseix, Jennifer Lu-Kuo, Thomas Ried, David Ward, Ilya Chumakov, Denis Le Paslier, Emmanuel Barillot and Daniel CohenCell 70: 1059-1068.[PDF]
Physical mapping of the human genome has until now been envisioned through single chromosome strategies. We demonstrate that by using large insert yeast artificial chromosomes (YACs) a whole genome approach becomes feasible. YACs (22,000) of 810 kb mean size (5 genome equivalents) have been fingerprinted to obtain individual patterns of restriction fragments detected by a LINE-1 (L1) probe. More than 1000 contigs were assembled. Ten randomly chosen contigs were validated by metaphase chromosome fluorescence in situ hybridization, as well as by analyzing the inter-Alu PCR patterns of their constituent YACs. We estimate that 15% to 20% of the human genome, mainly the L1-rich regions, is already covered with contigs larger than 3 Mb.
19 . Molecular basis of myotonic dystrophy: Expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family memberJ.David Brook, Mila E. McCurrach, Helen G. Harley, Alan J. Buckler, Deanna Church, Hiroyuki Aburatani, Kent Hunter, Vincent P. Stanton, Jean-Paul Thirion, Thomas Hudson, Robert Sohn, Boris Zemelman, Russell G. Snell, Shelley A. Rundle, Steve Crow, June Davies, Peggy Shelbourne, Jessica Buxton, Clare Jones, Vesa Juvonen, Keith Johnson, Peter S. Harper, Duncan J. Shaw and David E. HousmanCell 68: 799-808.[PDF]
Using positional cloning strategies, we have identified a CTG triplet repeat that undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population. PCR analysis of the interval containing this repeat indicates that unaffected individuals have between 5 and 27 copies. Myotonic dystrophy patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs. The CTG repeat is transcribed and is located in the 3′ untranslated region of an mRNA that is expressed in tissues affected by myotonic dystrophy. This mRNA encodes a polypeptide that is a member of the protein kinase family.
20 . In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epitheliumMelissa A. Rosenfeld, Kunihiko Yoshimura, Bruce C. Trapnell, Koichi Yoneyama, Eugene R. Rosenthal, Wilfried Dalemans, Masashi Fukayama, Joachim Bargon, Larue E. Stier, Leslie Stratford-Perricaudet, Michel Perricaudet, William B. Guggino, Andrea Pavirani, Jean-Pierre Lecocq and Ronald G. CrystalCell 68: 143-155.[PDF]
Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl− permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11–14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.
21 . Multipotent neural cell lines can engraft and participate in development of mouse cerebellumEvan Y. Snyder, David L. Deitcher, Christopher Walsh, Susan Arnold-Aldea, Erika A. Hartwieg and Constance L. CepkoCell 68: 33-51.[PDF]
Multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into murine cerebellar progenitor cells. When transplanted back into the cerebellum of newborn mice, these cells integrated into the cerebellum in a nontumorigenic, cytoarchitecturally appropriate manner. Cells from the same clonal line differentiated into neurons or glia in a manner appropriate to their site of engraftment. Engrafted cells, identified by lacZ expression and PCR-mediated detection of a unique sequence arrangement, could be identified in animals up to 22 months postengraftment. Electron microscopic and immunohistochemical analysis demonstrated that some engrafted cells were similar to host neurons and glia. Some transplant-derived neurons received appropriate synapses and formed normal intercellular contacts. These data indicate that generating immortalized cell lines for repair of, or transport of genes into, the CNS may be feasible. Such lines may also provide a model for commitment and differentiation of cerebellar progenitor cells.
22 . A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacteriumJosé Muñoz-Dorado, Sumiko Inouye and Masayori InouyeCell 67: 995-1006.[PDF]
PCR reactions were carried out on the genomic DNA of M. xanthus, a soil bacterium capable of differentiation to form fruiting bodies, using oligonucleotides representing highly conserved regions of eukaryotic protein serine/threonine kinases. A gene (pkn1) thus cloned contains an ORF of 693 amino acid residues whose amino-terminal domain shows significant sequence similarity with the catalytic domain of eukaryotic protein serine/threonine kinases. The pkn1 gene was overexpressed in E. coli, and the gene product has been found to be autophosphorylated at both serine and threonine residues. The expression of pkn1 is developmentally regulated to start immediately before spore formation. When pkn1 is deleted, differentiation starts prematurely, resulting in poor spore production. These results indicate that the protein serine/threonine kinase plays an important role in the onset of proper differentiation.
23 . CREM gene: Use of alternative DNA-binding domains generates multiple antagonists of cAMP-induced transcriptionNicholas S. Foulkes, Emiliana Borrelli and Paolo Sassone-CorsiCell 64: 739-749.[PDF]
We isolated a gene from a mouse pituitary cDNA library that encodes a protein highly homologous to nuclear factor CREB, an activator of cAMP-responsive promoter elements (CREs). We demonstrate that while CREB is expressed uniformly in several cell types, this gene, termed CREM, shows cell-specific expression. CREM has a remarkable organization, since downstream of the stop codon there is a second, out-of-frame DNA-binding domain. Using PCR and RNAase protection analysis, we have identified three mRNA isoforms that appear to be obtained by differential cell-specific splicing. Sequencing of the isoforms demonstrated alternative usage of the two DNA-binding domains. CREM proteins reveal the same efficiency and specificity of binding to CRE sequences as CREB, but in contrast to CREB, CREM acts as a down-regulator of cAMP-induced transcription.
24 . Scrambled exonsJanice M. Nigro, Kathleen R. Cho, Eric R. Fearon, Scott E. Kern, J.Michael Ruppert, Jonathan D. Oliner, Kenneth W. Kinzler and Bert VogelsteinCell 64: 607-613.[PDF]
Using a sensitive assay for RNA expression, we identified several abnormally spliced transcripts in which exons from a candidate tumor suppressor gene (DCC) were scrambled during the splicing process in vivo. Cloning and sequencing of PCR-amplified segments of the abnormally spliced transcripts showed that exons were joined accurately at consensus splice sites, but in an order different from that present in the primary transcript. Four scrambled transcripts were identified, each involving a different pair of exons. The scrambled transcripts were found at relatively low levels in a variety of normal and neoplastic cells of rodent and human origin, primarily in the nonpolyadenylated component of cytoplasmic RNA. These results demonstrate that the splicing process does not always pair sequential exons in the order predicted from their positions in genomic DNA, thus creating a novel type of RNA product.
25 . Retrotransposition of a mouse IAP sequence tagged with an indicator geneOdile Heldmann and Thierry HeidmannCell 64: 159-170.[PDF]
We have marked a cloned mouse IAP sequence with a neomycin-containing indicator gene whose expression is conditioned by passage of the transposon through an RNA intermediate. Transposition of the marked IAP introduced into tumor cells could be detected by simple selection of the cells in G418, at a frequency of 10−6 per cell per generation. Southern blot analysis and nucleotide sequencing after PCR amplification demonstrated “retrotransposition” of the marked element, with spllcing out of an Intron contained in the indicator gene, and retroviral-like reverse transcription and integration of the transposed IAPs, with 6 bp dupiications of the identified target sites. Transposition was found to be mutagenic for the element, as might be expected if the identified marked and endogenous IAP transcripts were coencapsidated into IAP particies as dimers.
26 . Activin can induce the formation of axial structures and is expressed in the hypoblast of the chickE. Mitrani, T. Ziv, G. Thomsen, Y. Shimoni, D.A. Melton and A. BrilCell 63: 495-501.[PDF]
We show that PIF/activin can induce the formation of axial structures including a full-length notochord, segmented somites, and a neural tube in isolated epiblasts from chick blastulae. Using degenerate PCR primers, we have cloned a fragment of the activin βB chain from chick hypobiast cDNA, and a fragment of the activin βA chain from chick genomic DNA. Furthermore, we show that in the chick, activin is transcribed precisely when axial mesoderm is being induced. Since exogenous PIF/activin can induce the formation of axial structures and since activin βB is transcribed at the time and place where the mesodermal axial structures are being induced, we propose that in the chick, activin B is the endogenous inducer of the body axis.
27 . A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutationsRichard M. Cawthon, Robert Weiss, Gangfeng Xu, David Viskochil, Melanie Culver, Jeff Stevens, Margaret Robertson, Diane Dunn, Ray Gesteland, Peter O'Connell and Ray WhiteCell 62: 193-201.[PDF]
Overlapping cDNA clones from the translocation break-point region (TBR) gene, recently discovered at the neurofibromatosis type 1 locus and found to be interrupted by deletions and a t(17;22) translocation, have been sequenced. A 4 kb sequence of the transcript of the TBR gene has been compared with sequences of genomic DNA, identifying a number of small exons. Identification of splice junctions and a large open reading frame indicates that the gene is oriented with its 5′ end toward the centromere, in opposition to the three known active genes in the region. PCR amplification of a subset of the exons, followed by electrophoresis of denatured product on native gels, identified six variant conformers specific to NF1 patients, indicating base pair changes in the gene. Sequencing revealed that one mutant allele contains a T→C transition changing a leucine to a proline; another NF1 allele harbors a C→T transition changing an arginine to a stop codon. These results establish the TBR gene as the NF1 gene and provide a description of a major segment of the gene.
28 . Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factorJamison Nourse, Julia D. Mellentin, Naomi Galili, Joyce Wilkinson, Eric Stanbridge, Stephen D. Smith and Michael L. ClearyCell 60: 535-545.[PDF]
The gene (E2A) for enhancer binding transcription factors E12 and E47 maps to the t(1;19) chromosomal translocation breakpoint in pre-B cell leukemias. Altered E2A transcripts lacking sequences coding for the helix-loop-helix DNA binding motif were detected in several t(1;19)-carrying cell lines. Fusion cDNAs that crossed the t(1;19) breakpoint were cloned and shown to code for an 85 kd protein consisting of the amino-terminal two-thirds of E2A fused to a chromosome 1-derived protein. The fusion protein has the features of a chimeric transcription factor in which the DNA binding domain of E2A is replaced by the putative DNA binding domain of a homeoprotein from chromosome 1 for which the name Prl (pre-B cell leukemia) is proposed. Identical E2A-prl mRNA junctions were detected by PCR in three t(1;19)-carrying cell lines, indicating that the fusion transcripts and predicted chimeric protein are a consistent feature of this translocation.
29 . Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolationsAndreas Meyerhans, Rémi Cheynier, Jan Albert, Martina Seth, Shirley Kwok, John Sninsky, Linda Morfeldt-Månson, Birgitta Asjö and Simon Wain-HobsonCell 58: 901-910.[PDF]
A genetic study has been made of the HIV tat gene from sequential HIV-1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for Iatently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.
1 . A Short Primer on RNAi: RNA-Directed RNA Polymerase Acts as a Key Catalyst
Kazuko NishikuraCell 107: 415-418.[Full Text] [PDF]
One of the many intriguing features of gene silencing by RNA interference is the apparent catalytic nature of the phenomenon. New biochemical and genetic evidence now shows that an RNA-directed RNA polymerase chain reaction, primed by siRNA, amplifies the interference caused by a small amount of “trigger” dsRNA.
2 . Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection
Peter M. Pryciak and Harold E. VarmusCell 69: 769-780.[PDF]
Integration of retroviral DNA can serve as a paradigm for cellular functions that are affected by the packaging of DNA into chromatin. We have used a novel polymerase chain reaction-based assay to survey DNA and chromatin for the precise distribution of many integration sites. Integration into naked DNA targets is nonuniform, implying a nucleotide sequence bias. In chromatin, integration occurs preferentially at postitions where the major groove is on the exposed face of the nucleosomal DNA helix, generating a 10 bp periodic spacing of preferred sites. Chromatin assembly enhances the reactivity of many sites, so that integration occurs most frequently at sites in nucleosomal, rather than nucleosome-free, regions of minichromosomes. In contrast, integration is prevented in a region occupied by a site-specific DNA-binding protein. Comparisons of integration events mediated by viral nucleoprotein complexes or by two different retroviral integrases show that the integration machinery also affects target site selection.
3 . Ubiquitous MyoD transcription at the midblastula transition precedes induction-dependent MyoD expression in presumptive mesoderm of X. laevis
Ralph A.W. Rupp and Harold WeintraubCell 65: 927-937.[PDF]
We have used a quantitative reverse transcription-polymerase chain reaction assay to detect MyoD mRNA during early embryonic development of Xenopus laevis. We find that during a short period of time following the midblastula transition MyoD becomes transcriptionally activated at a low level ubiquitously throughout the embryo. Restriction of MyoD expression to muscle precursor cells appears as a subsequent event, in which the process of mesoderm induction stabilizes transcription only in the marginal zone of the embryo, the presumptive mesoderm.
4 . Chimeric gRNA-mRNA molecules with oligo(U) tails covalently linked at sites of RNA editing suggest that U addition occurs by transesterification
Beat Blum, Nancy R. Sturm, Agda M. Simpson and Larry SimpsonCell 65: 543-550.[PDF]
Chimeric RNA molecules were detected by polymerase chain reaction ampllfication of kinetoplast RNA using a 3′ primer specific to mRNA and a 5′ primer specific to guide RNA (gRNA), and directly by Northern analysis. Covalent linkage of the 3′ oligo(U) tail of the gRNA to the mRNA occurs at editing sites. Chimeric molecules were isolated for NADH dehydrogenase subunit 7 and cytochrome oxidase subunits II and III. We propose that these molecules are intermediates in the editing process and that successive transesterifications resuit in the transfer of uridine residues from the gRNA 3′ oligo(U) tail to an editing site, with the number of uridine residues determined by base pairing with adenine and guanine “guide” nucleotides in the gRNA.
5 . A cyclin B homolog in S. cerevisiae: Chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis
Jayant B. Ghiara, Helena E. Richardson, Katsunori Sugimoto, Martha Henze, Daniel J. Lew, Curt Wittenberg and Steven I. ReedCell 65: 163-174.[PDF]
A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thoughtto be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.
6 . The recombination activating gene-1 (RAG-1) transcript is present in the murine central nervous system
Jerold J.M. Chun, David G. Schatz, Marjorie A. Oettinger, Rudolf Jaenisch and David BaltimoreCell 64: 189-200.[PDF]
The recombination activating genes, RAG-1 and RAG-2, are likely to encode components of the V(D)J site-specific recombination machinery. We report here the detection of low levels of the RAG-1 transcrlpt in the murlne central nervous system by polymerase chain reaction, In situ hybridization, and Northern blot analyses. In contrast, an authentic RAG-2 transcript could not be detected reproduclbly in the central nervous system. The RAG-1 transcript was found to be wide-spread in embryonic and postnatal neurons, with transcription being most apparent in regions of the postnatal brain with a high neuronal cell denslty (the cerebellum and the hippocampal formation). The results suggest that RAG-1 functions in neurons, where its role might be to recombine elements of the neuronal genome site-specifically, or to prevent detrimental alterations of the genome in these long-lived cells.
7 . Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from leishmania tarentolae mitochondria: RNA editing intermediates
Nancy R. Sturm and Larry SimpsonCell 61: 871-878.[PDF]
Partially edited mRNAs were selected by the polymerase chain reaction and sequenced. In the case of cytochrome b, 102 out of 106 clones displayed patterns of editing that were consistent with a strictly progressive 3′ to 5′ editing process, as predicted by the guide RNA model of RNA editing. In the case of cytochrome oxidase subunit III (COIII), 177 out of 304 clones displayed strictly progressive 3′ to 5′ patterns of editing. However, the remaining 127 COIII clones displayed unexpected patterns in which upstream editing preceded downstream editing, uridines were inserted at sites not normally edited, and purine residues were deleted. We suggest that many of these RNAs are produced by normal 3′ to 5′ editing of the COIII mRNA with incorrect guide RNA molecules.
8 . Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor
Hansruedi Loetscher, Yu-Ching E. Pan, Hans-Werner Lahm, Reiner Gentz, Manfred Brockhaus, Hisahiro Tabuchi and Werner LesslauerCell 61: 351-359.[PDF]
Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd are expressed at low levels by various cells. The 55 kd TNF receptor was purified from HL60 cells, and partial amino acid sequences were determined. Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction. The cDNA fragment as a probe identified several overlapping clones in a human placenta cDNA library. The open reading frame of the cDNA predicts a 455 amino acid TNF receptor protein with leader, extracellular, transmembrane, and intracellular domains. When expressed in COS-1 cells or in a baculovirus system, the cDNA conferred TNF binding properties comparable to the native receptor. Surprisingly, the 55 kd TNF receptor shows a high degree of sequence homology to the NGF receptor extracellular domain.
9 . HIV-1 entry into quiescent primary lymphocytes: Molecular analysis reveals a labile, latent viral structure
Jerome A. Zack, Salvatore J. Arrigo, Stacy R. Weitsman, Alan S. Go, Allyson Haislip and Irvin S.Y. ChenCell 61: 213-222.[PDF]
Productive infection of human T lymphocytes by HIV-1 is dependent upon proliferation of the infected cell. Nonproliferating quiescent T cells can be infected by HIV-1 and harbor the virus in an inactive state until subsequent mitogenic stimulation. We use a modification of the polymerase chain reaction method, which is both sensitive and quantitative, to demonstrate that HIV-1 DNA synthesis is initiated in infected quiescent T cells at levels comparable with those of activated T cells. However, unlike that of activated T cells, the viral genome is not completely reverse transcribed in quiescent cells. Although this viral DNA structure can persist in quiescent cells as a latent form, it is labile. We discuss the lability of this HIV-1 DNA structure in relation to a “self-restricting persistent infection” by HIV-1 and propose that this may explain the low percentage of infected cells in the circulation of AIDS patients.
10 . Detection of circular forms of eliminated DNA during macronuclear development in E. crassus
S.Lorraine Tausta and Lawrence A. KlobutcherCell 59: 1019-1026.[PDF]
Following their sexual cycle, hypotrichous ciliated protozoa transform a copy of a chromosomal micronucleus into a macronucleus containing small, linear DNA molecules. A frequent event during macronuclear development is the removal of short segments of DNA (internal eliminated sequences: IESs) by a process equivalent to DNA breakage and rejoining. In this study we used a polymerase chain reaction procedure to demonstrate that free circular forms of IESs are present in cells undergoing macronuclear development. Sequencing of the junctions of the free circular IESs suggests that they share 12 nucleotides with the macronuclear DNA molecules that are generated following IES removal. The results provide evidence that IESs are removed by an active DNA breakage and rejoining process, which may involve staggered cuts in the substrate DNA.
11 . Activation of immunoglobulin kappa gene rearrangement correlates with induction of germline kappa gene transcription
Mark S. Schlissel and David BaltimoreCell 58: 1001-1007.[PDF]
We have developed a sensitive polymerase chain reaction assay for measuring the fraction of rearranged immunoglobulin kappa genes in a cell population. Using this assay with Abelson virus-transformed murine pre-B cells, we have found that bacterial lipopolysacharide treatment, which activates transcription of the unrearranged kappa constant region gene, also activates kappa gene rearrangement. In addition, we have been able to detect kappa gene rearrangement in cell lines that do not produce a functional heavy chain gene product (mu protein). These results implicate transcription or transcription factor binding as a regulator of immunoglobulin gene rearrangement.
12 . Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations
Andreas Meyerhans, Rémi Cheynier, Jan Albert, Martina Seth, Shirley Kwok, John Sninsky, Linda Morfeldt-Månson, Birgitta Asjö and Simon Wain-HobsonCell 58: 901-910.[PDF]
A genetic study has been made of the HIV tat gene from sequential HIV-1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for Iatently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.
13 . Catalytic deficiency of human aldolase B in hereditary fructose intolerance caused by a common missense mutation
Nicholas C.P. Cross, Dean R. Tolan and Timothy M. CoxCell 53: 881-885.[PDF]
Hereditary fructose intolerance (HFI) is a human autosomal recessive disease caused by a deficiency of aldolase B that results in an inability to metabolize fructose and related sugars. We report here the first identification of a molecular lesion in the aldolase B gene of an affected individual whose defective protein has previously been characterized. The mutation is a G→C transversion in exon 5 that creates a new recognition site for the restriction enzyme Ahall and results in an amino acid substitution (Ala→Pro) at position 149 of the protein within a region critical for substrate binding. Utilizing this novel restriction site and the polymerase chain reaction, the patient was shown to be homozygous for the mutation. Three other HFI patients from pedigrees unrelated to this individual were found to have the same mutation: two were homozygous and one was heterozygous. We suggest that this genetic lesion is a prevailing cause of hereditary fructose intolerance.
14 . Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes
Concepcion Almoguera, Darryl Shibata, Kathleen Forrester, John Martin, Norman Arnheim and Manuel PeruchoCell 53: 549-554.[PDF]
Using in vitro gene amplification by the polymerase chain reaction (PCR) and mutation detection by the RNAase A mismatch cleavage method, we have examined, c-K-ras genes in human pancreatic carcinomas. We used frozen tumor specimens and single 5 μm sections from formalin-fixed, paraffin-embedded tumor tissue surgically removed or obtained at autopsy. Twenty-one out of 22 carcinmas of the exocrine pancreas contained c-K-ras genes with mutations at codon 12. In seven cases tested, the mutation was present in both primary tumors and their corresponding metastases. from the same cancer patients or in five gall bladder carcinomas. We conclude from these results that c-K-ras somatic mutational activation is a critical event in the oncogenesis of most, if not all, human cancers of the exocrine pancreas.
Symbiotic bacteria are important in animal hosts, but have been largely overlooked as they have proved difficult to culture in the laboratory. Approaches such as comparative genomics and real-time PCR have provided insights into the molecular mechanisms that underpin symbiont-host interactions. Studies on the heritable symbionts of insects have yielded valuable information about how bacteria infect host cells, avoid immune responses, and manipulate host physiology. Furthermore, some symbionts use many of the same mechanisms as pathogens to infect hosts and evade immune responses. Here we discuss what is currently known about the interactions between bacterial symbionts and their hosts.
2 . Loss of ARNT/HIF1β Mediates Altered Gene Expression and Pancreatic-Islet Dysfunction in Human Type 2 DiabetesJenny E. Gunton, Rohit N. Kulkarni, SunHee Yim, Terumasa Okada, Wayne J. Hawthorne, Yu-Hua Tseng, Russell S. Roberson, Camillo Ricordi, Philip J. O’Connell, Frank J. Gonzalez and C. Ronald KahnCell 122: 337-349.[Full Text] [PDF]
β cell dysfunction is a central component of the pathogenesis of type 2 diabetes. Using oligonucleotide microarrays and real-time PCR of pancreatic islets isolated from humans with type 2 diabetes versus normal glucose-tolerant controls, we identified multiple changes in expression of genes known to be important in β cell function, including major decreases in expression of HNF4α, insulin receptor, IRS2, Akt2, and several glucose-metabolic-pathway genes. There was also a 90% decrease in expression of the transcription factor ARNT. Reducing ARNT levels in Min6 cells with small interfering RNA (siRNA) resulted in markedly impaired glucose-stimulated insulin release and changes in gene expression similar to those in human type 2 islets. Likewise, β cell-specific ARNT knockout mice exhibited abnormal glucose tolerance, impaired insulin secretion, and changes in islet gene expression that mimicked those in human diabetic islets. Together, these data suggest an important role for decreased ARNT and altered gene expression in the impaired islet function of human type 2 diabetes.
3 . From Microbes to Prions: The Final Proof of the Prion HypothesisWen-Quan Zou and Pierluigi GambettiCell 121: 155-157.[Full Text] [PDF]
Much like the “microbe hypothesis” put forth over 150 years ago, the “prion hypothesis” can be definitely proven only if a prion disease is engendered in a natural host from an infectious prion produced in vitro. In this issue of Cell, Akman et al., 2002Abbott et al., 2000Castilla et al., 2005Blackburn, 1992 come very close to accomplishing this goal by producing a prion disease in a natural host from a prion entirely generated in vitro using a PCR-like amplification system.
4 . Microarray Identification of FMRP-Associated Brain mRNAs and Altered mRNA Translational Profiles in Fragile X SyndromeVictoria Brown, Peng Jin, Stephanie Ceman, Jennifer C. Darnell, William T. O'Donnell, Scott A. Tenenbaum, Xiaokui Jin, Yue Feng, Keith D. Wilkinson, Jack D. Keene, Robert B. Darnell and Stephen T. WarrenCell 107: 477-487.[Full Text] [PDF]
Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.
5 . RNAi as Random Degradative PCR: siRNA Primers Convert mRNA into dsRNAs that Are Degraded to Generate New siRNAsConcetta Lipardi, Qin Wei and Bruce M. PatersonCell 107: 297-307.[Full Text] [PDF]
In posttranscriptional gene silencing (PTGS), “quelling,” and RNA interference (RNAi), 21–25 nucleotide RNA fragments are produced from the initiating dsRNA. These short interfering RNAs (siRNAs) mediate RNAi by an unknown mechanism. Here, we show that GFP and Pp-Luc siRNAs, isolated from a protein complex in Drosophila embryo extract, target mRNA degradation in vitro. Most importantly, these siRNAs, as well as a synthetic 21-nucleotide duplex GFP siRNA, serve as primers to transform the target mRNA into dsRNA. The nascent dsRNA is degraded to eliminate the incorporated target mRNA while generating new siRNAs in a cycle of dsRNA synthesis and degradation. Evidence is presented that mRNA-dependent siRNA incorporation to form dsRNA is carried out by an RNA-dependent RNA polymerase activity (RdRP).
6 . Combinatorial Receptor Codes for OdorsBettina Malnic, Junzo Hirono, Takaaki Sato and Linda B Buck96: 713-723.[Full Text] [PDF]
The discriminatory capacity of the mammalian olfactory system is such that thousands of volatile chemicals are perceived as having distinct odors. Here we used a combination of calcium imaging and single-cell RT–PCR to identify odorant receptors (ORs) for odorants with related structures but varied odors. We found that one OR recognizes multiple odorants and that one odorant is recognized by multiple ORs, but that different odorants are recognized by different combinations of ORs. Thus, the olfactory system uses a combinatorial receptor coding scheme to encode odor identities. Our studies also indicate that slight alterations in an odorant, or a change in its concentration, can change its “code,” potentially explaining how such changes can alter perceived odor quality.
7 . Requirement for Specific Proteases in Cancer Cell Intravasation as Revealed by a Novel Semiquantitative PCR-Based AssayJ Kim, W Yu, K Kovalski and L Ossowski94: 353-362.[Full Text] [PDF]
Proteases are crucial for cancer metastasis, but due to lack of assays, their role in intravasation has not yet been tested. We have developed a human Alu sequence PCR-based assay to quantitate intravasated cells in an in vivo model. We demonstrated that metalloproteinases (MMPs), and most likely MMP-9, are required for intravasation by showing that marimastat, an inhibitor of MMPs, reduced intravasation by more than 90%, and that only tumor cell lines expressing MMP-9 intravasated. Cells with low surface urokinase plasminogen activator (uPA) and uPA receptor (uPAR) were also incapable of intravasation, despite the presence of high levels of MMP-9. We concluded that breaching of the vascular wall is a rate-limiting step for intravasation, and consequently for metastasis, and that cooperation between uPA/uPAR and MMP-9 is required to complete this step.
8 . Neandertal DNA Sequences and the Origin of Modern HumansMatthias Krings, Anne Stone, Ralf W Schmitz, Heike Krainitzki, Mark Stoneking and Svante Pääbo90: 19-30.[Full Text] [PDF]
DNA was extracted from the Neandertal-type specimen found in 1856 in western Germany. By sequencing clones from short overlapping PCR products, a hitherto unknown mitochondrial (mt) DNA sequence was determined. Multiple controls indicate that this sequence is endogenous to the fossil. Sequence comparisons with human mtDNA sequences, as well as phylogenetic analyses, show that the Neandertal sequence falls outside the variation of modern humans. Furthermore, the age of the common ancestor of the Neandertal and modern human mtDNAs is estimated to be four times greater than that of the common ancestor of human mtDNAs. This suggests that Neandertals went extinct without contributing mtDNA to modern humans.
9 . Altered Ca2+ Responses in Muscles with Combined Mitochondrial and Cytosolic Creatine Kinase DeficienciesKaren Steeghs, Ad Benders, Frank Oerlemans, Arnold de Haan, Arend Heerschap, Wim Ruitenbeek, Carolina Jost, Jan van Deursen, Benjamin Perryman, Dirk Pette, Marloes Brückwilder, Jolande Koudijs, Paul Jap, Jacques Veerkamp and Bé Wieringa89: 93-103.[Full Text] [PDF]
We have blocked creatine kinase (CK)-mediated phosphocreatine (PCr) ATP transphosphorylation in skeletal muscle by combining targeted mutations in the genes encoding mitochondrial and cytosolic CK in mice. Contrary to expectation, the PCr level was only marginally affected, but the compound was rendered metabolically inert. Mutant muscles in vivo showed significantly impaired tetanic force output, increased relaxation times, altered mitochondrial volume and location, and conspicuous tubular aggregates of sarcoplasmic reticulum membranes, as seen in myopathies with electrolyte disturbances. In depolarized myotubes cultured in vitro, CK absence influenced both the release and sequestration of Ca2+. Our data point to a direct link between the CK–PCr system and Ca2+-flux regulation during the excitation and relaxation phases of muscle contraction.
10 . Beyond PCR: Biotechnology as an Emerging CultureAdam Telerman and Robert B Amson86: 707-708.[Full Text] [PDF] 11 . The FHIT Gene at 3p14.2 Is Abnormal in Lung CancerGabriella Sozzi, Maria Luisa Veronese, Massimo Negrini, Raffaele Baffa, Maria Grazia Cotticelli, Hiroshi Inoue, Silvana Tornielli, Silvana Pilotti, Laura De Gregorio, Ugo Pastorino, Marco A Pierotti, Masataka Ohta, Kay Huebner and Carlo M Croce85: 17-26.[Full Text] [PDF]
To determine the role of the FHIT gene, which encompasses the fragile site at 3p14.2, we analyzed 59 tumors of the small cell and non-small cell type by reverse transcription of FHIT mRNA, followed by PCR amplification and sequencing of products. Allelic losses affecting the gene were evaluated by microsatellite polymorphism analysis and genomic alterations by hybridization using cDNA and genomic probes. Small cell lung tumors (80%) and non-small cell lung cancers (40%) showed abnormalities in RNA transcripts of FHIT, and 76% of the tumors exhibited loss of FHIT alleles. Abnormal lung tumor transcripts lack two or more exons of the FHIT gene. Small cell lung cancer tumors and cell lines were analyzed by Southern blotting and showed rearranged BamHI fragments. These data suggest a critical role of the FHIT gene in lung carcinogenesis.
12 . An in vitro assay for saccharomyces telomerase requires EST1Jing-Jer Lin and Virginia A. Zakian81: 1127-1135.[PDF]
Telomerase activity was demonstrated in cell-free extracts from S. cerevisiae through the use of a PCR-based assay. As expected, this activity was eliminated by RNase or phenol treatment of the extract and was dependent on dGTP and dTTP. Telomerase was not detected in extracts prepared from cells grown for ∼ 30 or more cell divisions in the absence of the EST1 product, Est1 p. TLC1 RNA, which determines the sequence of telomeric DNA in vivo, was present in normal amounts in est1Δ cells. Moreover, TLC1 RNA specifically precipitated with epitope-tagged Est1p. These data indicate that Estlp is either a subunit of yeast telomerase or an accessory protein associated with telomerase that is essential in vitro for its activity.
13 . A novel form of Epstein-Barr virus latency in normal B cells in vivoEmily M Miyashita, Bin Yang, Kitty M.C Lam, Dorothy H Crawford and David A Thorley-LawsonCell 80: 593-601.[PDF]
We have developed a PCR assay that can detect a single Epstein-Barr virus (EBV) genome in the presence of 106 uninfected cells. Using this assay, we demonstrate that EBV persists, in the peripheral blood of all seropositive individuals tested, in CD19+, CD23−, and CD80 (B7)− B cells. We further show that the virus in these cells is latent, but readily reactivated to produce infectious immortalizing virus; therefore, these cells represent a true site of latent persistence. EBV was not significantly detected in monocytes or T cells. The frequency of infected cells in nine healthy donors varied from 23 to 625 per 107 B cells, but was relatively stable for each individual over the course of 2 years. We conclude that the EBV-infected cells in vivo are B cells with a nonactivated phenotype. This represents a novel form of latency in normal B cells.
14 . The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophyNatalie Roy, Mani S Mahadevan, Michael McLean, Gary Shutter, Zahra Yaraghi, Reza Farahani, Stephen Baird, Anne Besner-Johnston, Charles Lefebvre, Xiaolin Kang, Maysoon Salih, Huguette Aubry, Katsuyuki Tamai, Xiaoping Guan, Panayiotis Ioannou, Thomas O Crawford, Pieter J de Jong, Linda Surh, Joh-E Ikeda, Robert G Korneluk and Alex MacKenzieCell 80: 167-178.[PDF]
The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR. analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.
15 . HIV and T cell expansion in splenic white pulps is accompanied by infiltration of HIV-specific cytotoxic T lymphocytesRémi Cheynier, Sven Henrichwark, Fabienne Hadida, Eric Pelletier, Eric Oksenhendler, Brigitte Autran and Simon Wain-HobsonCell 78: 373-387.[PDF]
Human immunodeficiency virus (HIV) replication and T cell proliferation were investigated in situ by a PCR-based analysis of individual microdissected splenic white pulps. Founder effects, revealed by an exquisite compartmentalization of HIV genotypes and T cells, indicated the recruitment of latently infected CD4+ T cells through highly localized antigen presentation rather than the infection of CD4+ T lymphoblasts by blood-borne virus or immune complexes. HIV-infected white pulps could be infiltrated by HIV-specific cytotoxic T lymphocytes, thereby implicating them in CD4+ T cell destruction in vivo. Together these data describe an iterative and deleterious mechanism of antigen-driven T cell recruitment and activation, as well as HIV replication and spread, with consequent destruction of the newly infected cells.
16 . Skeletal muscles of mice deficient in muscle creatine kinase lack burst activityJan van Deursen, Arend Heerschap, Frank Oerlemans, Wim Rultenbeek, Paul Jap, Henk ter Laak and Bé WieringaCell 74: 621-631.[PDF]
To understand the physiological role of the creatine kinase-phosphocreatine (CK-PCr) system in muscle bioenergetics, a null mutation of the muscle CK (M-CK) gene was introduced into the germline of mice. Mutant mice show no alterations in absolute muscle force, but lack the ability to perform burst activity. Their fast-twitch fibers have an increased intermyofibrillar mitochondrial volume and an increased glycogenolytic/glycolytic potential. PCr and ATP levels are normal in resting M-CK-deficient muscles, but rates of high energy phosphate exchange between PCr and ATP are at least 20-fold reduced. Strikingly, PCr levels decline normally during muscle exercise, suggesting that M-CK-mediated conversion is not the only route for PCr utilization in active muscle.
17 . Hotspots for unselected Ty1 transposition events on yeast chromosome III are near tRNA genes and LTR sequencesH. Ji, D.P. Moore, M.A. Blomberg, L.T. Braiterman, D.F. Voytas, G. Natsoulis and J.D. BoekeCell 73: 1007-1018.[PDF]
A collection of yeast strains bearing single marked Ty1 insertions on chromosome III was generated. Over 100 such insertions were physically mapped by pulsed-field gel electrophoresis. These insertions are very nonrandomly distributed. Thirty-two such insertions were cloned by the inverted PCR technique, and the flanking DNA sequences were determined. The sequenced insertions all fell within a few very limited regions of chromosome III. Most of these regions contained tRNA coding regions and/or LTRs of preexisting transposable elements. Open reading frames were disrupted at a far lower frequency than expected for random transposition. The results suggest that the Ty1 integration machinery can detect regions of the genome that may represent “safe havens” for insertion. These regions of the genome do not contain any special DNA sequences, nor do they behave as particularly good targets for Ty1 integration in vitro, suggesting that the targeted regions have special properties allowing specific recognition in vivo.
18 . Mapping the whole human genome by fingerprinting yeast artificial chromosomesChristine Bellanné-Chantelot, Bruno Lacroix, Pierre Ougen, Alain Billault, Sandrine Beaufils, Stéphane Bertrand, Isabelle Georges, Fabrice Glibert, Isabelle Gros, Georges Lucotte, Laurent Susini, Jean-Jacques Codani, Philippe Gesnouin, Stuart Pook, Guy Vaysseix, Jennifer Lu-Kuo, Thomas Ried, David Ward, Ilya Chumakov, Denis Le Paslier, Emmanuel Barillot and Daniel CohenCell 70: 1059-1068.[PDF]
Physical mapping of the human genome has until now been envisioned through single chromosome strategies. We demonstrate that by using large insert yeast artificial chromosomes (YACs) a whole genome approach becomes feasible. YACs (22,000) of 810 kb mean size (5 genome equivalents) have been fingerprinted to obtain individual patterns of restriction fragments detected by a LINE-1 (L1) probe. More than 1000 contigs were assembled. Ten randomly chosen contigs were validated by metaphase chromosome fluorescence in situ hybridization, as well as by analyzing the inter-Alu PCR patterns of their constituent YACs. We estimate that 15% to 20% of the human genome, mainly the L1-rich regions, is already covered with contigs larger than 3 Mb.
19 . Molecular basis of myotonic dystrophy: Expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family memberJ.David Brook, Mila E. McCurrach, Helen G. Harley, Alan J. Buckler, Deanna Church, Hiroyuki Aburatani, Kent Hunter, Vincent P. Stanton, Jean-Paul Thirion, Thomas Hudson, Robert Sohn, Boris Zemelman, Russell G. Snell, Shelley A. Rundle, Steve Crow, June Davies, Peggy Shelbourne, Jessica Buxton, Clare Jones, Vesa Juvonen, Keith Johnson, Peter S. Harper, Duncan J. Shaw and David E. HousmanCell 68: 799-808.[PDF]
Using positional cloning strategies, we have identified a CTG triplet repeat that undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population. PCR analysis of the interval containing this repeat indicates that unaffected individuals have between 5 and 27 copies. Myotonic dystrophy patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs. The CTG repeat is transcribed and is located in the 3′ untranslated region of an mRNA that is expressed in tissues affected by myotonic dystrophy. This mRNA encodes a polypeptide that is a member of the protein kinase family.
20 . In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epitheliumMelissa A. Rosenfeld, Kunihiko Yoshimura, Bruce C. Trapnell, Koichi Yoneyama, Eugene R. Rosenthal, Wilfried Dalemans, Masashi Fukayama, Joachim Bargon, Larue E. Stier, Leslie Stratford-Perricaudet, Michel Perricaudet, William B. Guggino, Andrea Pavirani, Jean-Pierre Lecocq and Ronald G. CrystalCell 68: 143-155.[PDF]
Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl− permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11–14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.
21 . Multipotent neural cell lines can engraft and participate in development of mouse cerebellumEvan Y. Snyder, David L. Deitcher, Christopher Walsh, Susan Arnold-Aldea, Erika A. Hartwieg and Constance L. CepkoCell 68: 33-51.[PDF]
Multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into murine cerebellar progenitor cells. When transplanted back into the cerebellum of newborn mice, these cells integrated into the cerebellum in a nontumorigenic, cytoarchitecturally appropriate manner. Cells from the same clonal line differentiated into neurons or glia in a manner appropriate to their site of engraftment. Engrafted cells, identified by lacZ expression and PCR-mediated detection of a unique sequence arrangement, could be identified in animals up to 22 months postengraftment. Electron microscopic and immunohistochemical analysis demonstrated that some engrafted cells were similar to host neurons and glia. Some transplant-derived neurons received appropriate synapses and formed normal intercellular contacts. These data indicate that generating immortalized cell lines for repair of, or transport of genes into, the CNS may be feasible. Such lines may also provide a model for commitment and differentiation of cerebellar progenitor cells.
22 . A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacteriumJosé Muñoz-Dorado, Sumiko Inouye and Masayori InouyeCell 67: 995-1006.[PDF]
PCR reactions were carried out on the genomic DNA of M. xanthus, a soil bacterium capable of differentiation to form fruiting bodies, using oligonucleotides representing highly conserved regions of eukaryotic protein serine/threonine kinases. A gene (pkn1) thus cloned contains an ORF of 693 amino acid residues whose amino-terminal domain shows significant sequence similarity with the catalytic domain of eukaryotic protein serine/threonine kinases. The pkn1 gene was overexpressed in E. coli, and the gene product has been found to be autophosphorylated at both serine and threonine residues. The expression of pkn1 is developmentally regulated to start immediately before spore formation. When pkn1 is deleted, differentiation starts prematurely, resulting in poor spore production. These results indicate that the protein serine/threonine kinase plays an important role in the onset of proper differentiation.
23 . CREM gene: Use of alternative DNA-binding domains generates multiple antagonists of cAMP-induced transcriptionNicholas S. Foulkes, Emiliana Borrelli and Paolo Sassone-CorsiCell 64: 739-749.[PDF]
We isolated a gene from a mouse pituitary cDNA library that encodes a protein highly homologous to nuclear factor CREB, an activator of cAMP-responsive promoter elements (CREs). We demonstrate that while CREB is expressed uniformly in several cell types, this gene, termed CREM, shows cell-specific expression. CREM has a remarkable organization, since downstream of the stop codon there is a second, out-of-frame DNA-binding domain. Using PCR and RNAase protection analysis, we have identified three mRNA isoforms that appear to be obtained by differential cell-specific splicing. Sequencing of the isoforms demonstrated alternative usage of the two DNA-binding domains. CREM proteins reveal the same efficiency and specificity of binding to CRE sequences as CREB, but in contrast to CREB, CREM acts as a down-regulator of cAMP-induced transcription.
24 . Scrambled exonsJanice M. Nigro, Kathleen R. Cho, Eric R. Fearon, Scott E. Kern, J.Michael Ruppert, Jonathan D. Oliner, Kenneth W. Kinzler and Bert VogelsteinCell 64: 607-613.[PDF]
Using a sensitive assay for RNA expression, we identified several abnormally spliced transcripts in which exons from a candidate tumor suppressor gene (DCC) were scrambled during the splicing process in vivo. Cloning and sequencing of PCR-amplified segments of the abnormally spliced transcripts showed that exons were joined accurately at consensus splice sites, but in an order different from that present in the primary transcript. Four scrambled transcripts were identified, each involving a different pair of exons. The scrambled transcripts were found at relatively low levels in a variety of normal and neoplastic cells of rodent and human origin, primarily in the nonpolyadenylated component of cytoplasmic RNA. These results demonstrate that the splicing process does not always pair sequential exons in the order predicted from their positions in genomic DNA, thus creating a novel type of RNA product.
25 . Retrotransposition of a mouse IAP sequence tagged with an indicator geneOdile Heldmann and Thierry HeidmannCell 64: 159-170.[PDF]
We have marked a cloned mouse IAP sequence with a neomycin-containing indicator gene whose expression is conditioned by passage of the transposon through an RNA intermediate. Transposition of the marked IAP introduced into tumor cells could be detected by simple selection of the cells in G418, at a frequency of 10−6 per cell per generation. Southern blot analysis and nucleotide sequencing after PCR amplification demonstrated “retrotransposition” of the marked element, with spllcing out of an Intron contained in the indicator gene, and retroviral-like reverse transcription and integration of the transposed IAPs, with 6 bp dupiications of the identified target sites. Transposition was found to be mutagenic for the element, as might be expected if the identified marked and endogenous IAP transcripts were coencapsidated into IAP particies as dimers.
26 . Activin can induce the formation of axial structures and is expressed in the hypoblast of the chickE. Mitrani, T. Ziv, G. Thomsen, Y. Shimoni, D.A. Melton and A. BrilCell 63: 495-501.[PDF]
We show that PIF/activin can induce the formation of axial structures including a full-length notochord, segmented somites, and a neural tube in isolated epiblasts from chick blastulae. Using degenerate PCR primers, we have cloned a fragment of the activin βB chain from chick hypobiast cDNA, and a fragment of the activin βA chain from chick genomic DNA. Furthermore, we show that in the chick, activin is transcribed precisely when axial mesoderm is being induced. Since exogenous PIF/activin can induce the formation of axial structures and since activin βB is transcribed at the time and place where the mesodermal axial structures are being induced, we propose that in the chick, activin B is the endogenous inducer of the body axis.
27 . A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutationsRichard M. Cawthon, Robert Weiss, Gangfeng Xu, David Viskochil, Melanie Culver, Jeff Stevens, Margaret Robertson, Diane Dunn, Ray Gesteland, Peter O'Connell and Ray WhiteCell 62: 193-201.[PDF]
Overlapping cDNA clones from the translocation break-point region (TBR) gene, recently discovered at the neurofibromatosis type 1 locus and found to be interrupted by deletions and a t(17;22) translocation, have been sequenced. A 4 kb sequence of the transcript of the TBR gene has been compared with sequences of genomic DNA, identifying a number of small exons. Identification of splice junctions and a large open reading frame indicates that the gene is oriented with its 5′ end toward the centromere, in opposition to the three known active genes in the region. PCR amplification of a subset of the exons, followed by electrophoresis of denatured product on native gels, identified six variant conformers specific to NF1 patients, indicating base pair changes in the gene. Sequencing revealed that one mutant allele contains a T→C transition changing a leucine to a proline; another NF1 allele harbors a C→T transition changing an arginine to a stop codon. These results establish the TBR gene as the NF1 gene and provide a description of a major segment of the gene.
28 . Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factorJamison Nourse, Julia D. Mellentin, Naomi Galili, Joyce Wilkinson, Eric Stanbridge, Stephen D. Smith and Michael L. ClearyCell 60: 535-545.[PDF]
The gene (E2A) for enhancer binding transcription factors E12 and E47 maps to the t(1;19) chromosomal translocation breakpoint in pre-B cell leukemias. Altered E2A transcripts lacking sequences coding for the helix-loop-helix DNA binding motif were detected in several t(1;19)-carrying cell lines. Fusion cDNAs that crossed the t(1;19) breakpoint were cloned and shown to code for an 85 kd protein consisting of the amino-terminal two-thirds of E2A fused to a chromosome 1-derived protein. The fusion protein has the features of a chimeric transcription factor in which the DNA binding domain of E2A is replaced by the putative DNA binding domain of a homeoprotein from chromosome 1 for which the name Prl (pre-B cell leukemia) is proposed. Identical E2A-prl mRNA junctions were detected by PCR in three t(1;19)-carrying cell lines, indicating that the fusion transcripts and predicted chimeric protein are a consistent feature of this translocation.
29 . Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolationsAndreas Meyerhans, Rémi Cheynier, Jan Albert, Martina Seth, Shirley Kwok, John Sninsky, Linda Morfeldt-Månson, Birgitta Asjö and Simon Wain-HobsonCell 58: 901-910.[PDF]
A genetic study has been made of the HIV tat gene from sequential HIV-1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for Iatently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.
1 . A Short Primer on RNAi: RNA-Directed RNA Polymerase Acts as a Key Catalyst
Kazuko NishikuraCell 107: 415-418.[Full Text] [PDF]
One of the many intriguing features of gene silencing by RNA interference is the apparent catalytic nature of the phenomenon. New biochemical and genetic evidence now shows that an RNA-directed RNA polymerase chain reaction, primed by siRNA, amplifies the interference caused by a small amount of “trigger” dsRNA.
2 . Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection
Peter M. Pryciak and Harold E. VarmusCell 69: 769-780.[PDF]
Integration of retroviral DNA can serve as a paradigm for cellular functions that are affected by the packaging of DNA into chromatin. We have used a novel polymerase chain reaction-based assay to survey DNA and chromatin for the precise distribution of many integration sites. Integration into naked DNA targets is nonuniform, implying a nucleotide sequence bias. In chromatin, integration occurs preferentially at postitions where the major groove is on the exposed face of the nucleosomal DNA helix, generating a 10 bp periodic spacing of preferred sites. Chromatin assembly enhances the reactivity of many sites, so that integration occurs most frequently at sites in nucleosomal, rather than nucleosome-free, regions of minichromosomes. In contrast, integration is prevented in a region occupied by a site-specific DNA-binding protein. Comparisons of integration events mediated by viral nucleoprotein complexes or by two different retroviral integrases show that the integration machinery also affects target site selection.
3 . Ubiquitous MyoD transcription at the midblastula transition precedes induction-dependent MyoD expression in presumptive mesoderm of X. laevis
Ralph A.W. Rupp and Harold WeintraubCell 65: 927-937.[PDF]
We have used a quantitative reverse transcription-polymerase chain reaction assay to detect MyoD mRNA during early embryonic development of Xenopus laevis. We find that during a short period of time following the midblastula transition MyoD becomes transcriptionally activated at a low level ubiquitously throughout the embryo. Restriction of MyoD expression to muscle precursor cells appears as a subsequent event, in which the process of mesoderm induction stabilizes transcription only in the marginal zone of the embryo, the presumptive mesoderm.
4 . Chimeric gRNA-mRNA molecules with oligo(U) tails covalently linked at sites of RNA editing suggest that U addition occurs by transesterification
Beat Blum, Nancy R. Sturm, Agda M. Simpson and Larry SimpsonCell 65: 543-550.[PDF]
Chimeric RNA molecules were detected by polymerase chain reaction ampllfication of kinetoplast RNA using a 3′ primer specific to mRNA and a 5′ primer specific to guide RNA (gRNA), and directly by Northern analysis. Covalent linkage of the 3′ oligo(U) tail of the gRNA to the mRNA occurs at editing sites. Chimeric molecules were isolated for NADH dehydrogenase subunit 7 and cytochrome oxidase subunits II and III. We propose that these molecules are intermediates in the editing process and that successive transesterifications resuit in the transfer of uridine residues from the gRNA 3′ oligo(U) tail to an editing site, with the number of uridine residues determined by base pairing with adenine and guanine “guide” nucleotides in the gRNA.
5 . A cyclin B homolog in S. cerevisiae: Chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis
Jayant B. Ghiara, Helena E. Richardson, Katsunori Sugimoto, Martha Henze, Daniel J. Lew, Curt Wittenberg and Steven I. ReedCell 65: 163-174.[PDF]
A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thoughtto be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.
6 . The recombination activating gene-1 (RAG-1) transcript is present in the murine central nervous system
Jerold J.M. Chun, David G. Schatz, Marjorie A. Oettinger, Rudolf Jaenisch and David BaltimoreCell 64: 189-200.[PDF]
The recombination activating genes, RAG-1 and RAG-2, are likely to encode components of the V(D)J site-specific recombination machinery. We report here the detection of low levels of the RAG-1 transcrlpt in the murlne central nervous system by polymerase chain reaction, In situ hybridization, and Northern blot analyses. In contrast, an authentic RAG-2 transcript could not be detected reproduclbly in the central nervous system. The RAG-1 transcript was found to be wide-spread in embryonic and postnatal neurons, with transcription being most apparent in regions of the postnatal brain with a high neuronal cell denslty (the cerebellum and the hippocampal formation). The results suggest that RAG-1 functions in neurons, where its role might be to recombine elements of the neuronal genome site-specifically, or to prevent detrimental alterations of the genome in these long-lived cells.
7 . Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from leishmania tarentolae mitochondria: RNA editing intermediates
Nancy R. Sturm and Larry SimpsonCell 61: 871-878.[PDF]
Partially edited mRNAs were selected by the polymerase chain reaction and sequenced. In the case of cytochrome b, 102 out of 106 clones displayed patterns of editing that were consistent with a strictly progressive 3′ to 5′ editing process, as predicted by the guide RNA model of RNA editing. In the case of cytochrome oxidase subunit III (COIII), 177 out of 304 clones displayed strictly progressive 3′ to 5′ patterns of editing. However, the remaining 127 COIII clones displayed unexpected patterns in which upstream editing preceded downstream editing, uridines were inserted at sites not normally edited, and purine residues were deleted. We suggest that many of these RNAs are produced by normal 3′ to 5′ editing of the COIII mRNA with incorrect guide RNA molecules.
8 . Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor
Hansruedi Loetscher, Yu-Ching E. Pan, Hans-Werner Lahm, Reiner Gentz, Manfred Brockhaus, Hisahiro Tabuchi and Werner LesslauerCell 61: 351-359.[PDF]
Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd are expressed at low levels by various cells. The 55 kd TNF receptor was purified from HL60 cells, and partial amino acid sequences were determined. Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction. The cDNA fragment as a probe identified several overlapping clones in a human placenta cDNA library. The open reading frame of the cDNA predicts a 455 amino acid TNF receptor protein with leader, extracellular, transmembrane, and intracellular domains. When expressed in COS-1 cells or in a baculovirus system, the cDNA conferred TNF binding properties comparable to the native receptor. Surprisingly, the 55 kd TNF receptor shows a high degree of sequence homology to the NGF receptor extracellular domain.
9 . HIV-1 entry into quiescent primary lymphocytes: Molecular analysis reveals a labile, latent viral structure
Jerome A. Zack, Salvatore J. Arrigo, Stacy R. Weitsman, Alan S. Go, Allyson Haislip and Irvin S.Y. ChenCell 61: 213-222.[PDF]
Productive infection of human T lymphocytes by HIV-1 is dependent upon proliferation of the infected cell. Nonproliferating quiescent T cells can be infected by HIV-1 and harbor the virus in an inactive state until subsequent mitogenic stimulation. We use a modification of the polymerase chain reaction method, which is both sensitive and quantitative, to demonstrate that HIV-1 DNA synthesis is initiated in infected quiescent T cells at levels comparable with those of activated T cells. However, unlike that of activated T cells, the viral genome is not completely reverse transcribed in quiescent cells. Although this viral DNA structure can persist in quiescent cells as a latent form, it is labile. We discuss the lability of this HIV-1 DNA structure in relation to a “self-restricting persistent infection” by HIV-1 and propose that this may explain the low percentage of infected cells in the circulation of AIDS patients.
10 . Detection of circular forms of eliminated DNA during macronuclear development in E. crassus
S.Lorraine Tausta and Lawrence A. KlobutcherCell 59: 1019-1026.[PDF]
Following their sexual cycle, hypotrichous ciliated protozoa transform a copy of a chromosomal micronucleus into a macronucleus containing small, linear DNA molecules. A frequent event during macronuclear development is the removal of short segments of DNA (internal eliminated sequences: IESs) by a process equivalent to DNA breakage and rejoining. In this study we used a polymerase chain reaction procedure to demonstrate that free circular forms of IESs are present in cells undergoing macronuclear development. Sequencing of the junctions of the free circular IESs suggests that they share 12 nucleotides with the macronuclear DNA molecules that are generated following IES removal. The results provide evidence that IESs are removed by an active DNA breakage and rejoining process, which may involve staggered cuts in the substrate DNA.
11 . Activation of immunoglobulin kappa gene rearrangement correlates with induction of germline kappa gene transcription
Mark S. Schlissel and David BaltimoreCell 58: 1001-1007.[PDF]
We have developed a sensitive polymerase chain reaction assay for measuring the fraction of rearranged immunoglobulin kappa genes in a cell population. Using this assay with Abelson virus-transformed murine pre-B cells, we have found that bacterial lipopolysacharide treatment, which activates transcription of the unrearranged kappa constant region gene, also activates kappa gene rearrangement. In addition, we have been able to detect kappa gene rearrangement in cell lines that do not produce a functional heavy chain gene product (mu protein). These results implicate transcription or transcription factor binding as a regulator of immunoglobulin gene rearrangement.
12 . Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations
Andreas Meyerhans, Rémi Cheynier, Jan Albert, Martina Seth, Shirley Kwok, John Sninsky, Linda Morfeldt-Månson, Birgitta Asjö and Simon Wain-HobsonCell 58: 901-910.[PDF]
A genetic study has been made of the HIV tat gene from sequential HIV-1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for Iatently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.
13 . Catalytic deficiency of human aldolase B in hereditary fructose intolerance caused by a common missense mutation
Nicholas C.P. Cross, Dean R. Tolan and Timothy M. CoxCell 53: 881-885.[PDF]
Hereditary fructose intolerance (HFI) is a human autosomal recessive disease caused by a deficiency of aldolase B that results in an inability to metabolize fructose and related sugars. We report here the first identification of a molecular lesion in the aldolase B gene of an affected individual whose defective protein has previously been characterized. The mutation is a G→C transversion in exon 5 that creates a new recognition site for the restriction enzyme Ahall and results in an amino acid substitution (Ala→Pro) at position 149 of the protein within a region critical for substrate binding. Utilizing this novel restriction site and the polymerase chain reaction, the patient was shown to be homozygous for the mutation. Three other HFI patients from pedigrees unrelated to this individual were found to have the same mutation: two were homozygous and one was heterozygous. We suggest that this genetic lesion is a prevailing cause of hereditary fructose intolerance.
14 . Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes
Concepcion Almoguera, Darryl Shibata, Kathleen Forrester, John Martin, Norman Arnheim and Manuel PeruchoCell 53: 549-554.[PDF]
Using in vitro gene amplification by the polymerase chain reaction (PCR) and mutation detection by the RNAase A mismatch cleavage method, we have examined, c-K-ras genes in human pancreatic carcinomas. We used frozen tumor specimens and single 5 μm sections from formalin-fixed, paraffin-embedded tumor tissue surgically removed or obtained at autopsy. Twenty-one out of 22 carcinmas of the exocrine pancreas contained c-K-ras genes with mutations at codon 12. In seven cases tested, the mutation was present in both primary tumors and their corresponding metastases. from the same cancer patients or in five gall bladder carcinomas. We conclude from these results that c-K-ras somatic mutational activation is a critical event in the oncogenesis of most, if not all, human cancers of the exocrine pancreas.
Important Publications at Nature
1. PCR Replicating success
Pete Moore
SUMMARY: PCR often gets taken for granted, but there are ways of making it faster, more accurate and easier to perform. Pete Moore investigates.
CONTEXT: As a means of rapidly copying a selected template sequence from a DNA mixture in vitro, PCR by itself and in combination with other techniques has found a vast range of applications. These range from sequence detection and isolation for...
Nature 435, 235 - 238 (11 May 2005), doi: 10.1038/435235a, Technology Feature
Full Text PDF Rights and permissions Save this link
2. Codon optimization to PCR
SUMMARY: Recent introductions include an array of heart disease genes.
CONTEXT: A2 readerBeckman Coulterhttp://www.beckmancoulter.comFluorescence-based CCD microplate reader The A2 is designed for automatic quantification of hundreds of protein analytes on the A2 microarray plate as part of Beckman Coulter's A2...
Nature 425, 540 - 540 (02 Oct 2003), doi: 10.1038/425540a, New on the Market
Full Text PDF Rights and permissions Save this link
3. Putting the C into PCR
SUMMARY: A round-up of thermocyclers and other PCR-related kit.
CONTEXT: Replication System and TSPNunchttp://www.nuncbrand.comSteel yourself With pins made of stainless steel, the Nunc Replication System is capable of transferring small inocula (1.0µl from liquid, 0.1µl from solid supports), resulting in...
Nature 419, 95 - 96 (05 Sep 2002), doi: 10.1038/419095a, New on the Market
Full Text PDF Rights and permissions Save this link
4. Turning the heat on PCR
SUMMARY: Thermocycling and PCR are the themes this week.
CONTEXT: Auto-LidApplied Biosystemshttp://www.appliedbiosystems.comCover-up for System 9700 thermal cyclers The Auto-Lid dual 384-well module for the GeneAMP PCR System 9700 thermal cycler is driven by a stepper motor that provides precise...
Nature 415, 818 - 818 (14 Feb 2002), doi: 10.1038/415818a, New on the Market
Full Text PDF Rights and permissions Save this link
5. PCR amplification of the Irish potato famine pathogen from historic specimens
Jean B. Ristaino, Carol T. Groves, Gregory R. Parra
SUMMARY: Late blight, caused by the oomycete plant pathogen Phytophthora infestans, is a devastating disease of potato and was responsible for epidemics that led to the Irish potato famine in 1845 (refs 1
CONTEXT: Late blight, caused by the oomycete plant pathogen Phytophthora infestans, is a devastating disease of potato and was responsible for epidemics that led to the Irish potato famine in 1845 (refs 1,2,3,4,5). Before the 1980s, worldwide...
Nature 411, 695 - 697 (07 Jun 2001), doi: 10.1038/35079606, Letter
Abstract Full Text PDF Rights and permissions Save this link
6. Patent ruling could cut PCR enzyme prices
Rex Dalton
CONTEXT: San Diego The European Patent Office (EPO) last week revoked a patent for an important thermally stable enzyme used in the polymerase chain reaction (PCR) process for DNA amplification. The decision brings with it the prospect that the...
Nature 411, 622 - 622 (07 Jun 2001), doi: 10.1038/35079739, News
Full Text PDF Rights and permissions Save this link
7. Promega and Roche take up battle over PCR patents
Rex Dalton
SUMMARY: SAN FRANCISCO The Swiss-based pharmaceutical company Hoffman-La Roche Inc. is facing a series of legal hearings over the validity of two key patents that it holds on the polymerase chain reaction
CONTEXT: San Francisco The Swiss-based pharmaceutical company Hoffman-LaRoche is facing a series of legal hearings over the validity of its two key patents on the polymerase chain reaction (PCR). The process will culminate in a trial in a federal...
Nature 404, 7 - 7 (02 Mar 2000), doi: 10.1038/35003722, News
Full Text PDF Rights and permissions Save this link
8. Simplified hot start PCR
David E. Birch, L. Kolmodin, J. Wong, G. A. Zangenberg, M. A. Zoccoli, N. McKinney, K. K. Y. Young
SUMMARY: The use of a thermally activated DNA polymerase PCR gives improved specificity, sensitivity and product yield without additives or extra process steps.
CONTEXT: HIGH background and low specific product yield can occur in a polymerase chain reaction (PCR) when reaction components are mixed at room temperature1"3. Reaction setup below the optimal primer annealing temperature (usually 50-65 °C)...
Nature 381, 445 - 446 (30 May 1996), doi: 10.1038/381445a0, Product Review
PDF Rights and permissions Save this link
9. PCR identification of black caviar
Rob DeSalle, Vadim J. Birstein
CONTEXT: SIR - Sturgeons (family Acipenseridae) and paddlefishes (family Polyodontidae), well known as producers of caviar, are threatened by unregulated overfishing, dams eliminating access to spawning grounds, and pollution1"3. Three species...
Nature 381, 197 - 198 (16 May 1996), doi: 10.1038/381197a0, Scientific Correspondence
PDF Rights and permissions Save this link
10. Patent on PCR enzymes may re-ignite old controversy
David Dickson
CONTEXT: London. Controversy over the patent rights to Taq polymerase, one of the basic thermostable enzymes used in the polymerase chain reaction (PCR) process, is likely to resurface following a decision by the European Patent Office (EPO) to...
Nature 372, 212 - 212 (17 Nov 1994), doi: 10.1038/372212a0, News
PDF Rights and permissions Save this link
11. The progeny of sexual PCR
George P. Smith
CONTEXT: FOR about a decade now, molecular biologists have been introducing random mutations into genes in order to probe structure-function relationships and to evolve new functions or improve old ones in vitro. The most popular methods include...
Nature 370, 324 - 325 (04 Aug 1994), doi: 10.1038/370324a0, News and Views
PDF Rights and permissions Save this link
12. Long PCR
Suzanne Cheng, Sheng-Yung Chang, Patti Gravitt, Richard Respess
SUMMARY: As increasingly longer DNA targets are amplified reliably, new applications for PCR are becoming possible.
CONTEXT: USE of the polymerase chain reaction (PCR) to amplify DNA sequences has been widespread in molecular genetics research, including genome mapping and sequencing studies' 3. However, robust amplification of targets greater than ˜5...
Nature 369, 684 - 685 (23 Jun 1994), doi: 10.1038/369684a0, Product Review
PDF Rights and permissions Save this link
13. Multi-target PCR analysis by capillary electrophoresis and laser-induced fluorescence
Wei Lu, Dai-Shu Han, Ju Yuan, Jean-Marie Andrieu
SUMMARY: Quantitative analysis of polymerase chain reaction (PCR) amplified HIV-1 DNA or cDNA fragments is attained using an automated system that combines capillary-gel electrophoresis (CGE) for high-eff
CONTEXT: IN many applications in molecular biology and biomedical research, quantitative detection of target sequences in native nucleic acids is frequently required. Although the polymerase chain reaction (PCR) technology has enabled significant...
Nature 368, 269 - 271 (17 Mar 1994), doi: 10.1038/368269a0, Product Review
PDF Rights and permissions Save this link
14. Dead Romanovs identified PCR
SUMMARY: Apart from the usual crop of gene assignments (for hereditary haemorrhagic telanglectasia, for example) this month's issue carries further the genetics of expanding repeating elements.
CONTEXT: THE use of DNA analysis for forensic purposes still resembles laboratory inves-tigation in that those responsible cannot behave as automata. So much is clear from the genetic identification of the remains, found in a shallow grave 35 km...
Nature 367, 580 - 580 (10 Feb 1994), doi: 10.1038/367580a0, Nature Genetics
PDF Rights and permissions Save this link
15. Quantitative PCR
Rudolf J. Wiesner, Bea Beinbrech, J. Caspar Rüegg
CONTEXT: SIR - In their Product Review1 on the competitive polymerase chain reaction (PCR), a technique that compares the accumulation of two products derived from a known amount of standard and the target, respectively, to quantitate the initial...
Nature 366, 416 - 416 (02 Dec 1993), doi: 10.1038/366416b0, Scientific Correspondence
PDF Rights and permissions Save this link
16. PCR primer
Diane Gershon
SUMMARY: This week's sampler includes a new kit for the rapid purification of polymerase chain reaction (PCR) products, a new matrix for isolating PCR-quality DNA and a kit for PCR amplification of the 5'
CONTEXT: THE trapezoidal tooth design of the TaperTooth comb from Gensura Laborato-ries creates 3- or 4-mm deep wells in stand-ard 4- or 5-mm thick analytical agarose gels (Reader Service No. 100). Gensura says that triangular-shaped agarose well...
Nature 365, 189 - 191 (09 Sep 1993), doi: 10.1038/365189a0, Product Review
PDF Rights and permissions Save this link
17. European patent for PCR enzyme clouded by Russian claim
David Dickson
CONTEXT: London. A Russian scientist whose research group was one of the first to isolate a thermostable enzyme from the bacterium Thermus aquaticus is claiming that his enzyme is identical to the widely used Taq DNA polymerase for which the US...
Nature 364, 2 - 2 (01 Jul 1993), doi: 10.1038/364002a0, News
PDF Rights and permissions Save this link
18. Licences sought from PCR users in Britain
David Dickson
CONTEXT: London. Genetic-screening services through-out Britain could have their costs increase significantly as a result of claims for royal-ties on one of the basic laboratory tech-niques used in diagnostic testing, polymerase chain reaction...
Nature 361, 291 - 291 (28 Jan 1993), doi: 10.1038/361291b0, News
PDF Rights and permissions Save this link
19. Competitive PCR
Paul D. Siebert, James W. Larrick
SUMMARY: Competitive reverse transcription-polymerase chain reaction (RT-PCR) can be used to obtain quantitative information of mRNA levels comparable to traditional RNA blot techniques, with the added ad
CONTEXT: THE combined use of reverse transcription followed by the polymerase chain reaction (RT-PCR) enables the amplification of individual RNA molecules. This method (recently reviewed in ref. 1) is variously termed RT-PCR2, RNA-PCR3, RNA...
Nature 359, 557 - 558 (08 Oct 1992), doi: 10.1038/359557a0, Product Review
PDF Rights and permissions Save this link
20. Excrement analysis by PCR
Matthias Höss, Michael Kohn, Svante Pääbo, Felix Knauer, Wolfgang Schröder
CONTEXT: SIR - Samples from endangered animals are hard to obtain for genetic analysis. To study a threatened bear population in the Pyrenees, Taberlet and Bouvet1 used hair collected from wire netting attached to trees on which the bears scratch...
Nature 359, 199 - 199 (17 Sep 1992), doi: 10.1038/359199a0, Scientific Correspondence
PDF Rights and permissions Save this link
21. Forensic use of PCR in Italy
ANGELO FIORI, VINCENZOL. PASCALI
CONTEXT: SIR - Following the admission of the use of evidence obtained through the polymerase chain reaction (PCR) in British courts1, attention has been drawn to the forensic use of PCR in Italy. Dallapiccola et al.2 reported that at the...
Nature 356, 471 - 471 (09 Apr 1992), doi: 10.1038/356471a0, Correspondence
PDF Rights and permissions Save this link
22. Roche cuts controversial PCR fees, testing limits
Christopher Anderson
CONTEXT: Researchers fought high prices, restrictions Firm promises reforms, may increase profits. UNDER pressure from researchers and diagnostic companies, Swiss-owned pharmaceutical company Hoffmann-La Roche last week agreed to lower its prices...
Nature 355, 379 - 379 (30 Jan 1992), doi: 10.1038/355379a0, News
PDF Rights and permissions Save this link
23. PCR DNA typing for forensics
BRUNO DALLAPICCOLA, GIUSEPPE NOVELLI, ALDO SPINELLA
CONTEXT: SIR - The first admission by a British court of forensic evidence using the polymerase chain reation (PCR)1' suggests that acceptance of this test could quickly become general outside the United States3. In actual casework, great care...
Nature 354, 179 - 179 (21 Nov 1991), doi: 10.1038/354179a0, Correspondence
PDF Rights and permissions Save this link
24. Cetus retains PCR patents
Elizabeth Schaefer
CONTEXT: San Francisco CETUS Corporation has finally nailed down its lucrative patent on the revolutionary polymerase chain reaction (PCR) technol-ogy. Last week, a US District Court upheld the validity of the patents granted to the biotechnology...
Nature 350, 6 - 6 (07 Mar 1991), doi: 10.1038/350006a0, News
PDF Rights and permissions Save this link
25. More light on PCR contamination
GOBINDA SARKAR, STEVE SOMMER
CONTEXT: SIR-In our recent Scientific Correspon-dence1 we showed that ultraviolet light was a potent inactivator of a 750-base pair segment of DNA when it contaminated reagents. All reagents, including Taq polymerase, could be decontaminated but,...
Nature 347, 340 - 341 (27 Sep 1990), doi: 10.1038/347340b0, Scientific Correspondence
PDF Rights and permissions Save this link
26. Improving PCR efficiency
B. FURRER, U. CANDRIAN, P. WIELAND, J. LÃœTHY
CONTEXT: SIR-The polymerase chain reaction (PCR) is a very sensitive in vitro DNA amplification method1. Therefore, carryover of even minute quantities of reaction products can lead to serious contamination problems and false-positive results....
Nature 346, 324 - 324 (26 Jul 1990), doi: 10.1038/346324b0, Scientific Correspondence
PDF Rights and permissions Save this link
27. PCR test for cystic fibrosis deletion
ANDREA BALLABIO, RICHARDA. GIBBS, C. THOMAS CASKEY
CONTEXT: SIR-Kerem et al.1 recently reported that approximately 70 per cent of the mutations in cystic fibrosis (CF) patients correspond to a specific deletion of 3 base pairs (bp) at amino-acid position 508 (AF508) of the putative product of the...
Nature 343, 220 - 220 (18 Jan 1990), doi: 10.1038/343220a0, Scientific Correspondence
PDF Rights and permissions Save this link
28. Shedding light on PCR contamination
GOBINDA SARKAR, STEVES SOMMER
CONTEXT: SIR-The most pernicious problem plaguing the widely used technique of polymerase chain reaction (PCR) is contamination of reagents with previously Effect of ultraviolet irradiation c 1 on PCR. A standard protocol6 was used to prepare...
Nature 343, 27 - 27 (04 Jan 1990), doi: 10.1038/343027a0, Scientific Correspondence
PDF Rights and permissions Save this link
29. PCR origins
JEFFREY S. PRICE
CONTEXT: SIR We noted with interest the short note, entitled "DNA amplification" (Nature 341, 570; 1989) which suggests that scientists who use PCR "dust off" the Journal of Molecular Biology from 1971, and read the paper by "H. Gobind Khorana...
Nature 342, 623 - 623 (07 Dec 1989), doi: 10.1038/342623b0, Scientific Correspondence
PDF Rights and permissions Save this link
30. PCR and more besides
T. L.
CONTEXT: 'LONG-awaited' is the tag publishers on occasion apply to their books that appear months, even years, after they were first announced - and, it turns out, to indifferent public reaction. That should not be the fate of the long-awaited...
Nature 341, 196 - 196 (21 Sep 1989), doi: 10.1038/341196b0, Book Review
PDF Rights and permissions Save this link
31. Avoiding false positives with PCR
S. Kwok, R. Higuchi
SUMMARY: The exquisite sensitivity of the polymerase chain reaction means DNA contamination can ruin an entire experiment. Tidiness and adherence to a strict set of protocols can avoid disaster.
CONTEXT: THE polymerase chain reaction (PCR)13 is a powerful, exquisitely sensitive34 technique with applications in many fields such as molecular biology, medical diagnostics, population genetics and forensic analysis. The use of specific DNA...
Nature 339, 237 - 238 (18 May 1989), doi: 10.1038/339237a0, Product Review
PDF Rights and permissions Save this link
32. Polymerase chain reaction reveals cloning artefacts
SVANTE PÄÄBO, ALLAN C. WILSON
CONTEXT: SIR-The recently developed polymerase chain reaction (PCR) makes possible the in vitro amplification of specific DNA segments bounded by an oligonucleotide primer on each strand1. During successive cycles of denaturation and extension of...
Nature 334, 387 - 388 (04 Aug 1988), doi: 10.1038/334387b0, Scientific Correspondence
PDF Rights and permissions Save this link
Pete Moore
SUMMARY: PCR often gets taken for granted, but there are ways of making it faster, more accurate and easier to perform. Pete Moore investigates.
CONTEXT: As a means of rapidly copying a selected template sequence from a DNA mixture in vitro, PCR by itself and in combination with other techniques has found a vast range of applications. These range from sequence detection and isolation for...
Nature 435, 235 - 238 (11 May 2005), doi: 10.1038/435235a, Technology Feature
Full Text PDF Rights and permissions Save this link
2. Codon optimization to PCR
SUMMARY: Recent introductions include an array of heart disease genes.
CONTEXT: A2 readerBeckman Coulterhttp://www.beckmancoulter.comFluorescence-based CCD microplate reader The A2 is designed for automatic quantification of hundreds of protein analytes on the A2 microarray plate as part of Beckman Coulter's A2...
Nature 425, 540 - 540 (02 Oct 2003), doi: 10.1038/425540a, New on the Market
Full Text PDF Rights and permissions Save this link
3. Putting the C into PCR
SUMMARY: A round-up of thermocyclers and other PCR-related kit.
CONTEXT: Replication System and TSPNunchttp://www.nuncbrand.comSteel yourself With pins made of stainless steel, the Nunc Replication System is capable of transferring small inocula (1.0µl from liquid, 0.1µl from solid supports), resulting in...
Nature 419, 95 - 96 (05 Sep 2002), doi: 10.1038/419095a, New on the Market
Full Text PDF Rights and permissions Save this link
4. Turning the heat on PCR
SUMMARY: Thermocycling and PCR are the themes this week.
CONTEXT: Auto-LidApplied Biosystemshttp://www.appliedbiosystems.comCover-up for System 9700 thermal cyclers The Auto-Lid dual 384-well module for the GeneAMP PCR System 9700 thermal cycler is driven by a stepper motor that provides precise...
Nature 415, 818 - 818 (14 Feb 2002), doi: 10.1038/415818a, New on the Market
Full Text PDF Rights and permissions Save this link
5. PCR amplification of the Irish potato famine pathogen from historic specimens
Jean B. Ristaino, Carol T. Groves, Gregory R. Parra
SUMMARY: Late blight, caused by the oomycete plant pathogen Phytophthora infestans, is a devastating disease of potato and was responsible for epidemics that led to the Irish potato famine in 1845 (refs 1
CONTEXT: Late blight, caused by the oomycete plant pathogen Phytophthora infestans, is a devastating disease of potato and was responsible for epidemics that led to the Irish potato famine in 1845 (refs 1,2,3,4,5). Before the 1980s, worldwide...
Nature 411, 695 - 697 (07 Jun 2001), doi: 10.1038/35079606, Letter
Abstract Full Text PDF Rights and permissions Save this link
6. Patent ruling could cut PCR enzyme prices
Rex Dalton
CONTEXT: San Diego The European Patent Office (EPO) last week revoked a patent for an important thermally stable enzyme used in the polymerase chain reaction (PCR) process for DNA amplification. The decision brings with it the prospect that the...
Nature 411, 622 - 622 (07 Jun 2001), doi: 10.1038/35079739, News
Full Text PDF Rights and permissions Save this link
7. Promega and Roche take up battle over PCR patents
Rex Dalton
SUMMARY: SAN FRANCISCO The Swiss-based pharmaceutical company Hoffman-La Roche Inc. is facing a series of legal hearings over the validity of two key patents that it holds on the polymerase chain reaction
CONTEXT: San Francisco The Swiss-based pharmaceutical company Hoffman-LaRoche is facing a series of legal hearings over the validity of its two key patents on the polymerase chain reaction (PCR). The process will culminate in a trial in a federal...
Nature 404, 7 - 7 (02 Mar 2000), doi: 10.1038/35003722, News
Full Text PDF Rights and permissions Save this link
8. Simplified hot start PCR
David E. Birch, L. Kolmodin, J. Wong, G. A. Zangenberg, M. A. Zoccoli, N. McKinney, K. K. Y. Young
SUMMARY: The use of a thermally activated DNA polymerase PCR gives improved specificity, sensitivity and product yield without additives or extra process steps.
CONTEXT: HIGH background and low specific product yield can occur in a polymerase chain reaction (PCR) when reaction components are mixed at room temperature1"3. Reaction setup below the optimal primer annealing temperature (usually 50-65 °C)...
Nature 381, 445 - 446 (30 May 1996), doi: 10.1038/381445a0, Product Review
PDF Rights and permissions Save this link
9. PCR identification of black caviar
Rob DeSalle, Vadim J. Birstein
CONTEXT: SIR - Sturgeons (family Acipenseridae) and paddlefishes (family Polyodontidae), well known as producers of caviar, are threatened by unregulated overfishing, dams eliminating access to spawning grounds, and pollution1"3. Three species...
Nature 381, 197 - 198 (16 May 1996), doi: 10.1038/381197a0, Scientific Correspondence
PDF Rights and permissions Save this link
10. Patent on PCR enzymes may re-ignite old controversy
David Dickson
CONTEXT: London. Controversy over the patent rights to Taq polymerase, one of the basic thermostable enzymes used in the polymerase chain reaction (PCR) process, is likely to resurface following a decision by the European Patent Office (EPO) to...
Nature 372, 212 - 212 (17 Nov 1994), doi: 10.1038/372212a0, News
PDF Rights and permissions Save this link
11. The progeny of sexual PCR
George P. Smith
CONTEXT: FOR about a decade now, molecular biologists have been introducing random mutations into genes in order to probe structure-function relationships and to evolve new functions or improve old ones in vitro. The most popular methods include...
Nature 370, 324 - 325 (04 Aug 1994), doi: 10.1038/370324a0, News and Views
PDF Rights and permissions Save this link
12. Long PCR
Suzanne Cheng, Sheng-Yung Chang, Patti Gravitt, Richard Respess
SUMMARY: As increasingly longer DNA targets are amplified reliably, new applications for PCR are becoming possible.
CONTEXT: USE of the polymerase chain reaction (PCR) to amplify DNA sequences has been widespread in molecular genetics research, including genome mapping and sequencing studies' 3. However, robust amplification of targets greater than ˜5...
Nature 369, 684 - 685 (23 Jun 1994), doi: 10.1038/369684a0, Product Review
PDF Rights and permissions Save this link
13. Multi-target PCR analysis by capillary electrophoresis and laser-induced fluorescence
Wei Lu, Dai-Shu Han, Ju Yuan, Jean-Marie Andrieu
SUMMARY: Quantitative analysis of polymerase chain reaction (PCR) amplified HIV-1 DNA or cDNA fragments is attained using an automated system that combines capillary-gel electrophoresis (CGE) for high-eff
CONTEXT: IN many applications in molecular biology and biomedical research, quantitative detection of target sequences in native nucleic acids is frequently required. Although the polymerase chain reaction (PCR) technology has enabled significant...
Nature 368, 269 - 271 (17 Mar 1994), doi: 10.1038/368269a0, Product Review
PDF Rights and permissions Save this link
14. Dead Romanovs identified PCR
SUMMARY: Apart from the usual crop of gene assignments (for hereditary haemorrhagic telanglectasia, for example) this month's issue carries further the genetics of expanding repeating elements.
CONTEXT: THE use of DNA analysis for forensic purposes still resembles laboratory inves-tigation in that those responsible cannot behave as automata. So much is clear from the genetic identification of the remains, found in a shallow grave 35 km...
Nature 367, 580 - 580 (10 Feb 1994), doi: 10.1038/367580a0, Nature Genetics
PDF Rights and permissions Save this link
15. Quantitative PCR
Rudolf J. Wiesner, Bea Beinbrech, J. Caspar Rüegg
CONTEXT: SIR - In their Product Review1 on the competitive polymerase chain reaction (PCR), a technique that compares the accumulation of two products derived from a known amount of standard and the target, respectively, to quantitate the initial...
Nature 366, 416 - 416 (02 Dec 1993), doi: 10.1038/366416b0, Scientific Correspondence
PDF Rights and permissions Save this link
16. PCR primer
Diane Gershon
SUMMARY: This week's sampler includes a new kit for the rapid purification of polymerase chain reaction (PCR) products, a new matrix for isolating PCR-quality DNA and a kit for PCR amplification of the 5'
CONTEXT: THE trapezoidal tooth design of the TaperTooth comb from Gensura Laborato-ries creates 3- or 4-mm deep wells in stand-ard 4- or 5-mm thick analytical agarose gels (Reader Service No. 100). Gensura says that triangular-shaped agarose well...
Nature 365, 189 - 191 (09 Sep 1993), doi: 10.1038/365189a0, Product Review
PDF Rights and permissions Save this link
17. European patent for PCR enzyme clouded by Russian claim
David Dickson
CONTEXT: London. A Russian scientist whose research group was one of the first to isolate a thermostable enzyme from the bacterium Thermus aquaticus is claiming that his enzyme is identical to the widely used Taq DNA polymerase for which the US...
Nature 364, 2 - 2 (01 Jul 1993), doi: 10.1038/364002a0, News
PDF Rights and permissions Save this link
18. Licences sought from PCR users in Britain
David Dickson
CONTEXT: London. Genetic-screening services through-out Britain could have their costs increase significantly as a result of claims for royal-ties on one of the basic laboratory tech-niques used in diagnostic testing, polymerase chain reaction...
Nature 361, 291 - 291 (28 Jan 1993), doi: 10.1038/361291b0, News
PDF Rights and permissions Save this link
19. Competitive PCR
Paul D. Siebert, James W. Larrick
SUMMARY: Competitive reverse transcription-polymerase chain reaction (RT-PCR) can be used to obtain quantitative information of mRNA levels comparable to traditional RNA blot techniques, with the added ad
CONTEXT: THE combined use of reverse transcription followed by the polymerase chain reaction (RT-PCR) enables the amplification of individual RNA molecules. This method (recently reviewed in ref. 1) is variously termed RT-PCR2, RNA-PCR3, RNA...
Nature 359, 557 - 558 (08 Oct 1992), doi: 10.1038/359557a0, Product Review
PDF Rights and permissions Save this link
20. Excrement analysis by PCR
Matthias Höss, Michael Kohn, Svante Pääbo, Felix Knauer, Wolfgang Schröder
CONTEXT: SIR - Samples from endangered animals are hard to obtain for genetic analysis. To study a threatened bear population in the Pyrenees, Taberlet and Bouvet1 used hair collected from wire netting attached to trees on which the bears scratch...
Nature 359, 199 - 199 (17 Sep 1992), doi: 10.1038/359199a0, Scientific Correspondence
PDF Rights and permissions Save this link
21. Forensic use of PCR in Italy
ANGELO FIORI, VINCENZOL. PASCALI
CONTEXT: SIR - Following the admission of the use of evidence obtained through the polymerase chain reaction (PCR) in British courts1, attention has been drawn to the forensic use of PCR in Italy. Dallapiccola et al.2 reported that at the...
Nature 356, 471 - 471 (09 Apr 1992), doi: 10.1038/356471a0, Correspondence
PDF Rights and permissions Save this link
22. Roche cuts controversial PCR fees, testing limits
Christopher Anderson
CONTEXT: Researchers fought high prices, restrictions Firm promises reforms, may increase profits. UNDER pressure from researchers and diagnostic companies, Swiss-owned pharmaceutical company Hoffmann-La Roche last week agreed to lower its prices...
Nature 355, 379 - 379 (30 Jan 1992), doi: 10.1038/355379a0, News
PDF Rights and permissions Save this link
23. PCR DNA typing for forensics
BRUNO DALLAPICCOLA, GIUSEPPE NOVELLI, ALDO SPINELLA
CONTEXT: SIR - The first admission by a British court of forensic evidence using the polymerase chain reation (PCR)1' suggests that acceptance of this test could quickly become general outside the United States3. In actual casework, great care...
Nature 354, 179 - 179 (21 Nov 1991), doi: 10.1038/354179a0, Correspondence
PDF Rights and permissions Save this link
24. Cetus retains PCR patents
Elizabeth Schaefer
CONTEXT: San Francisco CETUS Corporation has finally nailed down its lucrative patent on the revolutionary polymerase chain reaction (PCR) technol-ogy. Last week, a US District Court upheld the validity of the patents granted to the biotechnology...
Nature 350, 6 - 6 (07 Mar 1991), doi: 10.1038/350006a0, News
PDF Rights and permissions Save this link
25. More light on PCR contamination
GOBINDA SARKAR, STEVE SOMMER
CONTEXT: SIR-In our recent Scientific Correspon-dence1 we showed that ultraviolet light was a potent inactivator of a 750-base pair segment of DNA when it contaminated reagents. All reagents, including Taq polymerase, could be decontaminated but,...
Nature 347, 340 - 341 (27 Sep 1990), doi: 10.1038/347340b0, Scientific Correspondence
PDF Rights and permissions Save this link
26. Improving PCR efficiency
B. FURRER, U. CANDRIAN, P. WIELAND, J. LÃœTHY
CONTEXT: SIR-The polymerase chain reaction (PCR) is a very sensitive in vitro DNA amplification method1. Therefore, carryover of even minute quantities of reaction products can lead to serious contamination problems and false-positive results....
Nature 346, 324 - 324 (26 Jul 1990), doi: 10.1038/346324b0, Scientific Correspondence
PDF Rights and permissions Save this link
27. PCR test for cystic fibrosis deletion
ANDREA BALLABIO, RICHARDA. GIBBS, C. THOMAS CASKEY
CONTEXT: SIR-Kerem et al.1 recently reported that approximately 70 per cent of the mutations in cystic fibrosis (CF) patients correspond to a specific deletion of 3 base pairs (bp) at amino-acid position 508 (AF508) of the putative product of the...
Nature 343, 220 - 220 (18 Jan 1990), doi: 10.1038/343220a0, Scientific Correspondence
PDF Rights and permissions Save this link
28. Shedding light on PCR contamination
GOBINDA SARKAR, STEVES SOMMER
CONTEXT: SIR-The most pernicious problem plaguing the widely used technique of polymerase chain reaction (PCR) is contamination of reagents with previously Effect of ultraviolet irradiation c 1 on PCR. A standard protocol6 was used to prepare...
Nature 343, 27 - 27 (04 Jan 1990), doi: 10.1038/343027a0, Scientific Correspondence
PDF Rights and permissions Save this link
29. PCR origins
JEFFREY S. PRICE
CONTEXT: SIR We noted with interest the short note, entitled "DNA amplification" (Nature 341, 570; 1989) which suggests that scientists who use PCR "dust off" the Journal of Molecular Biology from 1971, and read the paper by "H. Gobind Khorana...
Nature 342, 623 - 623 (07 Dec 1989), doi: 10.1038/342623b0, Scientific Correspondence
PDF Rights and permissions Save this link
30. PCR and more besides
T. L.
CONTEXT: 'LONG-awaited' is the tag publishers on occasion apply to their books that appear months, even years, after they were first announced - and, it turns out, to indifferent public reaction. That should not be the fate of the long-awaited...
Nature 341, 196 - 196 (21 Sep 1989), doi: 10.1038/341196b0, Book Review
PDF Rights and permissions Save this link
31. Avoiding false positives with PCR
S. Kwok, R. Higuchi
SUMMARY: The exquisite sensitivity of the polymerase chain reaction means DNA contamination can ruin an entire experiment. Tidiness and adherence to a strict set of protocols can avoid disaster.
CONTEXT: THE polymerase chain reaction (PCR)13 is a powerful, exquisitely sensitive34 technique with applications in many fields such as molecular biology, medical diagnostics, population genetics and forensic analysis. The use of specific DNA...
Nature 339, 237 - 238 (18 May 1989), doi: 10.1038/339237a0, Product Review
PDF Rights and permissions Save this link
32. Polymerase chain reaction reveals cloning artefacts
SVANTE PÄÄBO, ALLAN C. WILSON
CONTEXT: SIR-The recently developed polymerase chain reaction (PCR) makes possible the in vitro amplification of specific DNA segments bounded by an oligonucleotide primer on each strand1. During successive cycles of denaturation and extension of...
Nature 334, 387 - 388 (04 Aug 1988), doi: 10.1038/334387b0, Scientific Correspondence
PDF Rights and permissions Save this link
Important Publications at Science
Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria
Elizabeth A. Ottesen, Jong Wook Hong, Stephen R. Quake, and Jared R. LeadbetterScience 1 December 2006 314: 1464-1467 [DOI: 10.1126/science.1131370] (in Reports)
......Reports MICROBIO Microfluidic Digital PCR Enables Multigene Analysis of Individual...microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different...catalyzing important activities in situ ( 1 ). PCR-based techniques that use single genes as......
Abstract » Full Text » PDF » Supporting Online Material »
DATABASE: Get Primed for PCR
Science 30 July 2004 305: 585 [DOI: 10.1126/science.305.5684.585b] (in NetWatch)
...netwatch NET LINK NW_MOLEC BIOL DATABASE: Get Primed for PCR Researchers use reverse transcription PCR to measure amounts of mRNA in a cell and gauge gene activity. Find the right primers for a particular......
Summary » PDF »
PCR in a Rayleigh-Bénard Convection Cell
Madhavi Krishnan, Victor M. Ugaz, and Mark A. BurnsScience 25 October 2002 298: 793 [DOI: 10.1126/science.298.5594.793] (in Brevia)
...Brevia BIOCHEM PCR in a Rayleigh-B e a nard Convection...convection to perform polymerase chain reaction (PCR) amplification of DNA inside a 35-Ml cylindrical...the diameter of the cavity. In the case of PCR, the required reaction efficiency constrains......
Full Text » PDF »
PCR:Roche Dealt a Setback on European Taq Patent
Robert F. ServiceScience 8 June 2001 292: 1815 [DOI: 10.1126/science.292.5523.1815a] (in News of the Week)
...n-week EUROPE NEWS SCI BUSINESS PCR: Roche Dealt a Setback on European Taq Patent Robert F. Service PCR. Roche dealt a setback on European Taq patent...Recombinant Proteins Taq Polymerase PCR:Roche Dealt a Setback on European Taq Patent......
Summary » Full Text »
PCR:Taq Polymerase Patent Ruled Invalid
Robert F. ServiceScience 17 December 1999 286: 2251-2253 [DOI: 10.1126/science.286.5448.2251b] (in News of the Week)
...PCR: Taq Polymerase Patent Ruled Invalid Robert F. Service...polymerase, is a crucial element of the polymerase chain reaction (PCR), the ubiquitous technique used to replicate snippets of DNA...Taq market, but Promega is challenging Roche's patents on PCR itself and may do the same with other Taq patents Roche holds......
Summary » Full Text »
INFECTIOUS DISEASE:PCR Detection of Bacteria in Seven Minutes
Phillip Belgrader, William Benett, Dean Hadley, James Richards, Paul Stratton, Raymond Mariella Jr., and Fred MilanovichScience 16 April 1999 284: 449-450 [DOI: 10.1126/science.284.5413.449] (in Tech.Sight)
...INFECTIOUS DISEASE: PCR Detection of Bacteria in Seven Minutes...bacterial tests use polymerase chain reaction (PCR) because of its demonstrated effectiveness...For field use, a variation of standard PCR called real-time PCR is the most practical......
Summary » Full Text »
SIGHTINGS:PCR on a Roller Coaster
Richard Peters and Robert SikorskiScience 19 June 1998 280: 1956 [DOI: 10.1126/science.280.5371.1956a] (in Tech.Sight)
...SIGHTINGS: PCR on a Roller Coaster Techwire Using...this topic. The polymerase chain reaction (PCR) has been a wildy successful and commonly...delays allowed the researchers to perform 20 PCR cycles in about 4 min and yield sample quality......
Full Text »
Performing PCR on a chip
Science 15 May 1998 280: 977 [DOI: 10.1126/science.280.5366.977c] (in This Week in Science)
...The three steps of the polymerase chain reaction (PCR), melting the double-stranded DNA, binding specific primers, and enzymatically extending the primers, have been automated on a......
Summary »
Chemical Amplification: Continuous-Flow PCR on a Chip
Martin U. Kopp, Andrew J. de Mello, and Andreas ManzScience 15 May 1998 280: 1046-1048 [DOI: 10.1126/science.280.5366.1046] (in Reports)
...Chemical Amplification: Continuous-Flow PCR on a Chip Martin U. Kopp...to perform the polymerase chain reaction (PCR) in continuous flow at high speed. The device...independent of input concentration. A 20-cycle PCR amplification of a 176-base pair fragment......
Abstract » Full Text » PDF »
SCIENCE AND COMMERCE:PCR Patent Tangle Slows Quick Assay of HIV Levels
Jon CohenScience 6 June 1997 276: 1488-1491 [DOI: 10.1126/science.276.5318.1488a] (in News & Comment)
...SCIENCE AND COMMERCE: PCR Patent Tangle Slows Quick Assay of HIV Levels...rights to the polymerase chain reaction (PCR), the basic analytic technique on which...Systems Inc., which holds the patent on PCR, PE Applied Biosystems cannot promote diagnostic......
Summary » Full Text »
Book ReviewsMaking PCR and Exquisite Specificity, reviewed by S. Hilgartner * Chondrules and the Protoplanetary Disk, H. Y. McSween Jr. * Biotic Recovery from Mass Extinction Events, J. H. Lipps * Vignette * Books Received
Science 8 November 1996 274: 934-936 [DOI: 10.1126/science.274.5289.934] (in Departments)
...Making PCR. A Story of Biotechnology. PAUL RABINOW. University...antibodies and the polymerase chain reaction (PCR) are widely regarded as among the most important...enjoy a vast market, and the patent rights to PCR sold in 1989 for a reported $300 million......
PDF »
Scientists named in PCR suit
M BarinagaScience 2 June 1995 268: 1273 [DOI: 10.1126/science.7761847] (in Articles)
......Race-Twice Rachel Nowak Scientists named in PCR suit. News EC 2.7.7.- Taq Polymerase...Rachel Nowak BIOTECH PATENTS Scientists Named in PCR Suit Ifyour lab uses the polymerase chain reac-tion (PCR), yourname may have come up in a San Francisco......
PDF »
PCR products and CITES
M JonesScience 23 December 1994 266: 1930 [DOI: 10.1126/science.7801116] (in Articles)
...articles PCR products and CITES M Jones PCR products and CITES. Comment Letter 9007-49-2 DNA Science...org (for submitting classi-fied advertisements) 1930 LETTERS PCR Products and CITES Brian Bowen and John Avise's letter, "Con......
PDF »
Conservation research and the legal status of PCR products
BW Bowen and JC AviseScience 4 November 1994 266: 713 [DOI: 10.1126/science.7973619] (in Articles)
......Conservation research and the legal status of PCR products BW Bowen JC Avise Conservation research and the legal status of PCR products. Letter 9007-49-2 DNA Science...Conservation Research and the Legal Status of PCR Products The challenge of obtaining biological......
PDF »
Advanced PCR
Mary Ann D. BrowScience 5 August 1994 265: 817-819 [DOI: 10.1126/science.265.5173.817-a] (in Articles)
......of Science Article Article Advanced PCR Mary Ann D. Brow Third Wave Technologies...Madison, WI 53711-5368, USA Advanced PCR. JOURNAL ARTICLE Modern Cosmology and...Washington, Seattle, WA 98195, USA a Advanced PCR The Polymerase Chain Reaction. KARY B. MULLIS......
PDF »
'Long PCR' leaps into larger DNA sequences
J CohenScience 18 March 1994 263: 1564-1565 [DOI: 10.1126/science.8128242] (in Articles)
...articles 'Long PCR' leaps into larger DNA sequences J Cohen 'Long PCR' leaps into larger DNA sequences. News 0 DNA Primers...business," he says.-Faye Flam MOLECULAR BIOLOGY 'Long PCR' Leaps Into Larger DNA Sequences On Christmas Eve 1992......
References » PDF »
Hantavirus outbreak yields to PCR
E MarshallScience 5 November 1993 262: 832-836 [DOI: 10.1126/science.8235602] (in Articles)
...articles Hantavirus outbreak yields to PCR E Marshall Hantavirus outbreak yields to PCR. News Science. 1993 Nov 5;262(5135):850-1...ICVVP~~~ a UIIIFEII Hantavirus Outbreak Yields to PCR Researchers have yet to isolate the virus that......
References » PDF »
PCR enzyme. The more you use, the cheaper it gets
P AldhousScience 6 August 1993 261: 678 [DOI: 10.1126/science.8342033] (in Articles)
...articles PCR enzyme. The more you use, the cheaper it gets P Aldhous PCR enzyme. The more you use, the cheaper it gets. News...economics Switzerland Taq Polymerase United States PCR ENZYME The More You Use, the Cheaper It Gets Big users......
PDF »
Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry
BK Patterson, M Till, P Otto, C Goolsby, MR Furtado, LJ McBride, and SM WolinskyScience 14 May 1993 260: 976-979 [DOI: 10.1126/science.8493534] (in Articles)
......and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry...and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry...and Messenger RNA in Individual Cells by PCR-Driven in Situ Hybridization and Flow Cytometry......
Abstract » References » PDF »
Immuno-PCR with a commercially available avidin system
V Ruzicka, W Marz, A Russ, and W GrossScience 30 April 1993 260: 698-699 [DOI: 10.1126/science.8480182] (in Articles)
...articles Immuno-PCR with a commercially available avidin system...Ruzicka W Marz A Russ W Gross Immuno-PCR with a commercially available avidin system...GTCCCCAGTCATCAGCTCCTG-3' and 5'-AGAGTTGCATGCCATGGTCCC-3'). PCR reac-tions (1 00-pI) were done with biotinylated......
References » PDF »
PCR enzyme patent challenged
P AldhousScience 23 April 1993 260: 486 [DOI: 10.1126/science.8475381] (in Articles)
......s Very Own Red Scare Wayne Biddle PCR enzyme patent challenged. News EC 2...biography ofvon Braun. INTELLECTUAL PROPERTY PCR Enzyme Patent Challenged Thesparkshave begun...make use of the polymerase chain reaction (PCR)-the gene-amplifying technique that's one......
PDF »
PCR Enzyme Patent Challenged
Peter AldhousScience 23 April 1993 260: 486 [DOI: 10.1126/science.260.5107.486-a] (in Articles)
......Advancement of Science Article Article PCR Enzyme Patent Challenged Peter Aldhous...biography ofvon Braun. INTELLECTUAL PROPERTY PCR Enzyme Patent Challenged Thesparkshave begun...make use of the polymerase chain reaction (PCR)-the gene-amplifying technique that's one......
PDF »
High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR
M Piatak, Jr, MS Saag, LC Yang, SJ Clark, JC Kappes, KC Luk, BH Hahn, GM Shaw, and JD LifsonScience 19 March 1993 259: 1749-1754 [DOI: 10.1126/science.8096089] (in Articles)
......stages of infection determined by competitive PCR M Piatak Jr MS Saag LC Yang SJ Clark...competitive polymerase chain reaction (QC-PCR) methods were used to quantify virion-associated...therapy. Plasma virus levels determined by QC-PCR correlated with, but exceeded by an average......
Abstract » References » PDF »
Roche gets tough on illicit sales of PCR reagent
P AldhousScience 4 December 1992 258: 1572-1573 [DOI: 10.1126/science.1455243] (in Articles)
...articles Roche gets tough on illicit sales of PCR reagent P Aldhous Roche gets tough on illicit sales of PCR reagent. News 0 Indicators and Reagents...REACrION Roche Gets Tough on Illicit Sales of PCR Reagent PCRusers beware. Ifyou have been......
References » PDF »
Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates
T Sano, CL Smith, and CR CantorScience 2 October 1992 258: 120-122 [DOI: 10.1126/science.1439758] (in Articles)
...articles Immuno-PCR: very sensitive antigen detection by means...immuno-polymerase chain reaction (immuno-PCR), was developed in which a specific DNA...segment of the attached DNA was amplified by PCR. Analysis of the PCR products by agarose......
Abstract » References » PDF »
PCR Regulations
Science 21 February 1992 255: 927 [DOI: 10.1126/science.255.5047.927-c] (in Articles)
......Advancement of Science ARTICLE ARTICLE PCR Regulations Parting blast from AIDS official...teachersinyourchildren's science classrooms. PCR Regulations Following up on its promise...the use ofthe polymerase chain reac-tion (PCR)(Science,31 January, p. 528), Hoffmann-La......
PDF »
PCR amplification of specific alleles
SS SommerScience 31 January 1992 255: 514 [DOI: 10.1126/science.1736349] (in Articles)
...articles PCR amplification of specific alleles SS Sommer PCR amplification of specific alleles. Comment Letter...genetics Polymerase Chain Reaction Restriction Mapping PCR Amplification of Specific Alleles Rick Weiss' Research......
PDF »
Roche eases PCR restrictions
M HoffmanScience 31 January 1992 255: 528 [DOI: 10.1126/science.1736354] (in Articles)
...articles Roche eases PCR restrictions M Hoffman Roche eases PCR restrictions. News Animals DNA Fingerprinting methods...on IL-2 were largely right. a RICHARD STONE Roche Eases PCR Restrictions When the powerful gene amplification tech-nique......
PDF »
Hoffmann—La Roche's PCR Push
ANN GIBBONSScience 9 August 1991 253: 627 [DOI: 10.1126/science.253.5020.627] (in Articles)
......for the Advancement of Science ARTICLE ARTICLE Hoffmann-La Roche's PCR Push ANN GIBBONS Hoffmann--La Roche's PCR Push. JOURNAL ARTICLE Hoffmann-La Roche's PCR Push While the polymerase chain reaction (PCR) has long been the darling......
PDF »
Identification of a peptide specific for Aplysia sensory neurons by PCR-based differential screening
JF Brunet, E Shapiro, SA Foster, ER Kandel, and Y IinoScience 10 May 1991 252: 856-859 [DOI: 10.1126/science.1840700] (in Articles)
......specific for Aplysia sensory neurons by PCR-based differential screening JF Brunet...specific for Aplysia sensory neurons by PCR-based differential screening. In order...Peptide Specific forAplysia Sensory Neurons by PCR-Based Differential Screening JEAN-FRAN0ois......
Abstract » References » PDF »
And the winner: Cetus does own PCR
M BarinagaScience 8 March 1991 251: 1174 [DOI: 10.1126/science.2006407] (in Articles)
...articles And the winner: Cetus does own PCR M Barinaga And the winner: Cetus does own PCR. News Biotechnology Drug Industry Patents...ANNE SIMON MOFFAT And the Winner: Cetus Does Own PCR Round 1 in a David and Goliath struggle between......
References » PDF »
Biotech nightmare: does Cetus own PCR?
M BarinagaScience 15 February 1991 251: 739-740 [DOI: 10.1126/science.1990435] (in Articles)
...articles Biotech nightmare: does Cetus own PCR? M Barinaga Biotech nightmare: does Cetus own PCR? News Biotechnology economics legislation...United States Biotech Nightmare: Does Cetus Own PCR? The biotech company is locked in combat with......
References » PDF »
PCR analysis of DNA from multiple sclerosis patients for the presence of HTLV-I
CHARLES R.M BANGHAM, SIMON NIGHTINGALE, J.K CRUICKSHANK, and SUSAN DAENKEScience 10 November 1989 246: 821 [DOI: 10.1126/science.2683085] (in Articles)
...articles PCR analysis of DNA from multiple sclerosis patients...NIGHTINGALE J.K CRUICKSHANK SUSAN DAENKE PCR analysis of DNA from multiple sclerosis patients...microbiology Polymerase Chain Reaction ___"Ill W PCR Analysis of DNA from Multiple Sclerosis Patients......
PDF »
In Reply: PCR Analysis of DNA from Multiple Sclerosis Patients for the Presence of HTLV-I
E. Premkuwar ReddyScience 10 November 1989 246: 823-824 [DOI: 10.1126/science.246.4931.823] (in Articles)
......Science Article Article In Reply: PCR Analysis of DNA from Multiple Sclerosis...Philadelphia, PA 19104-4268 In Reply: PCR Analysis of DNA from Multiple Sclerosis...Boston, MA 02115 peripheral blood MNCs. PCR reactions were done in a 50-Il volume with......
References » PDF »
In Reply: PCR Analysis of DNA from Multiple Sclerosis Patients for the Presence of HTLV-I
Jennifer H. Richardson, Kai W. Wucherpfenning, N. Endo, Peter Rudge, Angus G. Dalgleish, and David A. HaflerScience 10 November 1989 246: 821-823 [DOI: 10.1126/science.246.4931.821-a] (in Articles)
......Science Article Article In Reply: PCR Analysis of DNA from Multiple Sclerosis...Medical School, Boston, MA 02115 ___"Ill W PCR Analysis of DNA from Multiple Sclerosis...amplifica-tion by the polymerase chain reaction (PCR) of HTLV-I (4) or a related retrovirus......
PDF »
Genomic sequencing and methylation analysis by ligation mediated PCR
GP Pfeifer, SD Steigerwald, PR Mueller, B Wold, and AD RiggsScience 10 November 1989 246: 810-813 [DOI: 10.1126/science.2814502] (in Articles)
......methylation analysis by ligation mediated PCR GP Pfeifer SD Steigerwald PR Mueller...ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible...methylation analysis by ligation mediated PCR. Genomic sequencing permits studies of......
Abstract » References » PDF »
In vivo footprinting of a muscle specific enhancer by ligation mediated PCR
PR Mueller and B WoldScience 10 November 1989 246: 780-786 [DOI: 10.1126/science.2814500] (in Articles)
......muscle specific enhancer by ligation mediated PCR PR Mueller B Wold Division of Biology...newly developed polymerase chain reaction (PCR) footprinting procedure. This ligation mediated, single-sided PCR technique permits the exponential amplification......
Polymerase Chain Reaction: Promega Wins Round in Fight Over Taq
Marcia BarinagaScience 23 August 1996 273: 1039-0 [DOI: 10.1126/science.273.5278.1039a] (in News & Comment)
......legislation & jurisprudence Patents Polymerase Chain Reaction Taq Polymerase POLYMERASE CHAIN REACTION Promega Wins Round in Fight Over Taq...the popular technique known as the polymerase chain reaction (PCR)-took a new turn in a San Francisco......
Summary » PDF »
Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction
P Liang and AB PardeeScience 14 August 1992 257: 967-971 [DOI: 10.1126/science.1354393] (in Articles)
......messenger RNA by means of the polymerase chain reaction P Liang AB Pardee...mRNAs) by means of the polymerase chain reaction. The key element is to...messenger RNA by means of the polymerase chain reaction. Effective methods are......
Abstract » References » PDF »
Recent advances in the polymerase chain reaction
HA Erlich, D Gelfand, and JJ SninskyScience 21 June 1991 252: 1643-1651 [DOI: 10.1126/science.2047872] (in Articles)
......Recent advances in the polymerase chain reaction HA Erlich D Gelfand...Emeryville, CA 94608. The polymerase chain reaction (PCR) has dramatically...Recent advances in the polymerase chain reaction. The polymerase chain......
Abstract » References » PDF »
Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain
EY Loh, JF Elliott, S Cwirla, LL Lanier, and MM DavisScience 13 January 1989 243: 217-220 [DOI: 10.1126/science.2463672] (in Articles)
...articles Polymerase chain reaction with single-sided specificity...CA 94305-5402. In the polymerase chain reaction (PCR), two specific oligonucleotide...novel technique, anchored polymerase chain reaction (A-PCR), was devised that......
Abstract » References » PDF »
Elizabeth A. Ottesen, Jong Wook Hong, Stephen R. Quake, and Jared R. LeadbetterScience 1 December 2006 314: 1464-1467 [DOI: 10.1126/science.1131370] (in Reports)
......Reports MICROBIO Microfluidic Digital PCR Enables Multigene Analysis of Individual...microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different...catalyzing important activities in situ ( 1 ). PCR-based techniques that use single genes as......
Abstract » Full Text » PDF » Supporting Online Material »
DATABASE: Get Primed for PCR
Science 30 July 2004 305: 585 [DOI: 10.1126/science.305.5684.585b] (in NetWatch)
...netwatch NET LINK NW_MOLEC BIOL DATABASE: Get Primed for PCR Researchers use reverse transcription PCR to measure amounts of mRNA in a cell and gauge gene activity. Find the right primers for a particular......
Summary » PDF »
PCR in a Rayleigh-Bénard Convection Cell
Madhavi Krishnan, Victor M. Ugaz, and Mark A. BurnsScience 25 October 2002 298: 793 [DOI: 10.1126/science.298.5594.793] (in Brevia)
...Brevia BIOCHEM PCR in a Rayleigh-B e a nard Convection...convection to perform polymerase chain reaction (PCR) amplification of DNA inside a 35-Ml cylindrical...the diameter of the cavity. In the case of PCR, the required reaction efficiency constrains......
Full Text » PDF »
PCR:Roche Dealt a Setback on European Taq Patent
Robert F. ServiceScience 8 June 2001 292: 1815 [DOI: 10.1126/science.292.5523.1815a] (in News of the Week)
...n-week EUROPE NEWS SCI BUSINESS PCR: Roche Dealt a Setback on European Taq Patent Robert F. Service PCR. Roche dealt a setback on European Taq patent...Recombinant Proteins Taq Polymerase PCR:Roche Dealt a Setback on European Taq Patent......
Summary » Full Text »
PCR:Taq Polymerase Patent Ruled Invalid
Robert F. ServiceScience 17 December 1999 286: 2251-2253 [DOI: 10.1126/science.286.5448.2251b] (in News of the Week)
...PCR: Taq Polymerase Patent Ruled Invalid Robert F. Service...polymerase, is a crucial element of the polymerase chain reaction (PCR), the ubiquitous technique used to replicate snippets of DNA...Taq market, but Promega is challenging Roche's patents on PCR itself and may do the same with other Taq patents Roche holds......
Summary » Full Text »
INFECTIOUS DISEASE:PCR Detection of Bacteria in Seven Minutes
Phillip Belgrader, William Benett, Dean Hadley, James Richards, Paul Stratton, Raymond Mariella Jr., and Fred MilanovichScience 16 April 1999 284: 449-450 [DOI: 10.1126/science.284.5413.449] (in Tech.Sight)
...INFECTIOUS DISEASE: PCR Detection of Bacteria in Seven Minutes...bacterial tests use polymerase chain reaction (PCR) because of its demonstrated effectiveness...For field use, a variation of standard PCR called real-time PCR is the most practical......
Summary » Full Text »
SIGHTINGS:PCR on a Roller Coaster
Richard Peters and Robert SikorskiScience 19 June 1998 280: 1956 [DOI: 10.1126/science.280.5371.1956a] (in Tech.Sight)
...SIGHTINGS: PCR on a Roller Coaster Techwire Using...this topic. The polymerase chain reaction (PCR) has been a wildy successful and commonly...delays allowed the researchers to perform 20 PCR cycles in about 4 min and yield sample quality......
Full Text »
Performing PCR on a chip
Science 15 May 1998 280: 977 [DOI: 10.1126/science.280.5366.977c] (in This Week in Science)
...The three steps of the polymerase chain reaction (PCR), melting the double-stranded DNA, binding specific primers, and enzymatically extending the primers, have been automated on a......
Summary »
Chemical Amplification: Continuous-Flow PCR on a Chip
Martin U. Kopp, Andrew J. de Mello, and Andreas ManzScience 15 May 1998 280: 1046-1048 [DOI: 10.1126/science.280.5366.1046] (in Reports)
...Chemical Amplification: Continuous-Flow PCR on a Chip Martin U. Kopp...to perform the polymerase chain reaction (PCR) in continuous flow at high speed. The device...independent of input concentration. A 20-cycle PCR amplification of a 176-base pair fragment......
Abstract » Full Text » PDF »
SCIENCE AND COMMERCE:PCR Patent Tangle Slows Quick Assay of HIV Levels
Jon CohenScience 6 June 1997 276: 1488-1491 [DOI: 10.1126/science.276.5318.1488a] (in News & Comment)
...SCIENCE AND COMMERCE: PCR Patent Tangle Slows Quick Assay of HIV Levels...rights to the polymerase chain reaction (PCR), the basic analytic technique on which...Systems Inc., which holds the patent on PCR, PE Applied Biosystems cannot promote diagnostic......
Summary » Full Text »
Book ReviewsMaking PCR and Exquisite Specificity, reviewed by S. Hilgartner * Chondrules and the Protoplanetary Disk, H. Y. McSween Jr. * Biotic Recovery from Mass Extinction Events, J. H. Lipps * Vignette * Books Received
Science 8 November 1996 274: 934-936 [DOI: 10.1126/science.274.5289.934] (in Departments)
...Making PCR. A Story of Biotechnology. PAUL RABINOW. University...antibodies and the polymerase chain reaction (PCR) are widely regarded as among the most important...enjoy a vast market, and the patent rights to PCR sold in 1989 for a reported $300 million......
PDF »
Scientists named in PCR suit
M BarinagaScience 2 June 1995 268: 1273 [DOI: 10.1126/science.7761847] (in Articles)
......Race-Twice Rachel Nowak Scientists named in PCR suit. News EC 2.7.7.- Taq Polymerase...Rachel Nowak BIOTECH PATENTS Scientists Named in PCR Suit Ifyour lab uses the polymerase chain reac-tion (PCR), yourname may have come up in a San Francisco......
PDF »
PCR products and CITES
M JonesScience 23 December 1994 266: 1930 [DOI: 10.1126/science.7801116] (in Articles)
...articles PCR products and CITES M Jones PCR products and CITES. Comment Letter 9007-49-2 DNA Science...org (for submitting classi-fied advertisements) 1930 LETTERS PCR Products and CITES Brian Bowen and John Avise's letter, "Con......
PDF »
Conservation research and the legal status of PCR products
BW Bowen and JC AviseScience 4 November 1994 266: 713 [DOI: 10.1126/science.7973619] (in Articles)
......Conservation research and the legal status of PCR products BW Bowen JC Avise Conservation research and the legal status of PCR products. Letter 9007-49-2 DNA Science...Conservation Research and the Legal Status of PCR Products The challenge of obtaining biological......
PDF »
Advanced PCR
Mary Ann D. BrowScience 5 August 1994 265: 817-819 [DOI: 10.1126/science.265.5173.817-a] (in Articles)
......of Science Article Article Advanced PCR Mary Ann D. Brow Third Wave Technologies...Madison, WI 53711-5368, USA Advanced PCR. JOURNAL ARTICLE Modern Cosmology and...Washington, Seattle, WA 98195, USA a Advanced PCR The Polymerase Chain Reaction. KARY B. MULLIS......
PDF »
'Long PCR' leaps into larger DNA sequences
J CohenScience 18 March 1994 263: 1564-1565 [DOI: 10.1126/science.8128242] (in Articles)
...articles 'Long PCR' leaps into larger DNA sequences J Cohen 'Long PCR' leaps into larger DNA sequences. News 0 DNA Primers...business," he says.-Faye Flam MOLECULAR BIOLOGY 'Long PCR' Leaps Into Larger DNA Sequences On Christmas Eve 1992......
References » PDF »
Hantavirus outbreak yields to PCR
E MarshallScience 5 November 1993 262: 832-836 [DOI: 10.1126/science.8235602] (in Articles)
...articles Hantavirus outbreak yields to PCR E Marshall Hantavirus outbreak yields to PCR. News Science. 1993 Nov 5;262(5135):850-1...ICVVP~~~ a UIIIFEII Hantavirus Outbreak Yields to PCR Researchers have yet to isolate the virus that......
References » PDF »
PCR enzyme. The more you use, the cheaper it gets
P AldhousScience 6 August 1993 261: 678 [DOI: 10.1126/science.8342033] (in Articles)
...articles PCR enzyme. The more you use, the cheaper it gets P Aldhous PCR enzyme. The more you use, the cheaper it gets. News...economics Switzerland Taq Polymerase United States PCR ENZYME The More You Use, the Cheaper It Gets Big users......
PDF »
Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry
BK Patterson, M Till, P Otto, C Goolsby, MR Furtado, LJ McBride, and SM WolinskyScience 14 May 1993 260: 976-979 [DOI: 10.1126/science.8493534] (in Articles)
......and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry...and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry...and Messenger RNA in Individual Cells by PCR-Driven in Situ Hybridization and Flow Cytometry......
Abstract » References » PDF »
Immuno-PCR with a commercially available avidin system
V Ruzicka, W Marz, A Russ, and W GrossScience 30 April 1993 260: 698-699 [DOI: 10.1126/science.8480182] (in Articles)
...articles Immuno-PCR with a commercially available avidin system...Ruzicka W Marz A Russ W Gross Immuno-PCR with a commercially available avidin system...GTCCCCAGTCATCAGCTCCTG-3' and 5'-AGAGTTGCATGCCATGGTCCC-3'). PCR reac-tions (1 00-pI) were done with biotinylated......
References » PDF »
PCR enzyme patent challenged
P AldhousScience 23 April 1993 260: 486 [DOI: 10.1126/science.8475381] (in Articles)
......s Very Own Red Scare Wayne Biddle PCR enzyme patent challenged. News EC 2...biography ofvon Braun. INTELLECTUAL PROPERTY PCR Enzyme Patent Challenged Thesparkshave begun...make use of the polymerase chain reaction (PCR)-the gene-amplifying technique that's one......
PDF »
PCR Enzyme Patent Challenged
Peter AldhousScience 23 April 1993 260: 486 [DOI: 10.1126/science.260.5107.486-a] (in Articles)
......Advancement of Science Article Article PCR Enzyme Patent Challenged Peter Aldhous...biography ofvon Braun. INTELLECTUAL PROPERTY PCR Enzyme Patent Challenged Thesparkshave begun...make use of the polymerase chain reaction (PCR)-the gene-amplifying technique that's one......
PDF »
High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR
M Piatak, Jr, MS Saag, LC Yang, SJ Clark, JC Kappes, KC Luk, BH Hahn, GM Shaw, and JD LifsonScience 19 March 1993 259: 1749-1754 [DOI: 10.1126/science.8096089] (in Articles)
......stages of infection determined by competitive PCR M Piatak Jr MS Saag LC Yang SJ Clark...competitive polymerase chain reaction (QC-PCR) methods were used to quantify virion-associated...therapy. Plasma virus levels determined by QC-PCR correlated with, but exceeded by an average......
Abstract » References » PDF »
Roche gets tough on illicit sales of PCR reagent
P AldhousScience 4 December 1992 258: 1572-1573 [DOI: 10.1126/science.1455243] (in Articles)
...articles Roche gets tough on illicit sales of PCR reagent P Aldhous Roche gets tough on illicit sales of PCR reagent. News 0 Indicators and Reagents...REACrION Roche Gets Tough on Illicit Sales of PCR Reagent PCRusers beware. Ifyou have been......
References » PDF »
Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates
T Sano, CL Smith, and CR CantorScience 2 October 1992 258: 120-122 [DOI: 10.1126/science.1439758] (in Articles)
...articles Immuno-PCR: very sensitive antigen detection by means...immuno-polymerase chain reaction (immuno-PCR), was developed in which a specific DNA...segment of the attached DNA was amplified by PCR. Analysis of the PCR products by agarose......
Abstract » References » PDF »
PCR Regulations
Science 21 February 1992 255: 927 [DOI: 10.1126/science.255.5047.927-c] (in Articles)
......Advancement of Science ARTICLE ARTICLE PCR Regulations Parting blast from AIDS official...teachersinyourchildren's science classrooms. PCR Regulations Following up on its promise...the use ofthe polymerase chain reac-tion (PCR)(Science,31 January, p. 528), Hoffmann-La......
PDF »
PCR amplification of specific alleles
SS SommerScience 31 January 1992 255: 514 [DOI: 10.1126/science.1736349] (in Articles)
...articles PCR amplification of specific alleles SS Sommer PCR amplification of specific alleles. Comment Letter...genetics Polymerase Chain Reaction Restriction Mapping PCR Amplification of Specific Alleles Rick Weiss' Research......
PDF »
Roche eases PCR restrictions
M HoffmanScience 31 January 1992 255: 528 [DOI: 10.1126/science.1736354] (in Articles)
...articles Roche eases PCR restrictions M Hoffman Roche eases PCR restrictions. News Animals DNA Fingerprinting methods...on IL-2 were largely right. a RICHARD STONE Roche Eases PCR Restrictions When the powerful gene amplification tech-nique......
PDF »
Hoffmann—La Roche's PCR Push
ANN GIBBONSScience 9 August 1991 253: 627 [DOI: 10.1126/science.253.5020.627] (in Articles)
......for the Advancement of Science ARTICLE ARTICLE Hoffmann-La Roche's PCR Push ANN GIBBONS Hoffmann--La Roche's PCR Push. JOURNAL ARTICLE Hoffmann-La Roche's PCR Push While the polymerase chain reaction (PCR) has long been the darling......
PDF »
Identification of a peptide specific for Aplysia sensory neurons by PCR-based differential screening
JF Brunet, E Shapiro, SA Foster, ER Kandel, and Y IinoScience 10 May 1991 252: 856-859 [DOI: 10.1126/science.1840700] (in Articles)
......specific for Aplysia sensory neurons by PCR-based differential screening JF Brunet...specific for Aplysia sensory neurons by PCR-based differential screening. In order...Peptide Specific forAplysia Sensory Neurons by PCR-Based Differential Screening JEAN-FRAN0ois......
Abstract » References » PDF »
And the winner: Cetus does own PCR
M BarinagaScience 8 March 1991 251: 1174 [DOI: 10.1126/science.2006407] (in Articles)
...articles And the winner: Cetus does own PCR M Barinaga And the winner: Cetus does own PCR. News Biotechnology Drug Industry Patents...ANNE SIMON MOFFAT And the Winner: Cetus Does Own PCR Round 1 in a David and Goliath struggle between......
References » PDF »
Biotech nightmare: does Cetus own PCR?
M BarinagaScience 15 February 1991 251: 739-740 [DOI: 10.1126/science.1990435] (in Articles)
...articles Biotech nightmare: does Cetus own PCR? M Barinaga Biotech nightmare: does Cetus own PCR? News Biotechnology economics legislation...United States Biotech Nightmare: Does Cetus Own PCR? The biotech company is locked in combat with......
References » PDF »
PCR analysis of DNA from multiple sclerosis patients for the presence of HTLV-I
CHARLES R.M BANGHAM, SIMON NIGHTINGALE, J.K CRUICKSHANK, and SUSAN DAENKEScience 10 November 1989 246: 821 [DOI: 10.1126/science.2683085] (in Articles)
...articles PCR analysis of DNA from multiple sclerosis patients...NIGHTINGALE J.K CRUICKSHANK SUSAN DAENKE PCR analysis of DNA from multiple sclerosis patients...microbiology Polymerase Chain Reaction ___"Ill W PCR Analysis of DNA from Multiple Sclerosis Patients......
PDF »
In Reply: PCR Analysis of DNA from Multiple Sclerosis Patients for the Presence of HTLV-I
E. Premkuwar ReddyScience 10 November 1989 246: 823-824 [DOI: 10.1126/science.246.4931.823] (in Articles)
......Science Article Article In Reply: PCR Analysis of DNA from Multiple Sclerosis...Philadelphia, PA 19104-4268 In Reply: PCR Analysis of DNA from Multiple Sclerosis...Boston, MA 02115 peripheral blood MNCs. PCR reactions were done in a 50-Il volume with......
References » PDF »
In Reply: PCR Analysis of DNA from Multiple Sclerosis Patients for the Presence of HTLV-I
Jennifer H. Richardson, Kai W. Wucherpfenning, N. Endo, Peter Rudge, Angus G. Dalgleish, and David A. HaflerScience 10 November 1989 246: 821-823 [DOI: 10.1126/science.246.4931.821-a] (in Articles)
......Science Article Article In Reply: PCR Analysis of DNA from Multiple Sclerosis...Medical School, Boston, MA 02115 ___"Ill W PCR Analysis of DNA from Multiple Sclerosis...amplifica-tion by the polymerase chain reaction (PCR) of HTLV-I (4) or a related retrovirus......
PDF »
Genomic sequencing and methylation analysis by ligation mediated PCR
GP Pfeifer, SD Steigerwald, PR Mueller, B Wold, and AD RiggsScience 10 November 1989 246: 810-813 [DOI: 10.1126/science.2814502] (in Articles)
......methylation analysis by ligation mediated PCR GP Pfeifer SD Steigerwald PR Mueller...ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible...methylation analysis by ligation mediated PCR. Genomic sequencing permits studies of......
Abstract » References » PDF »
In vivo footprinting of a muscle specific enhancer by ligation mediated PCR
PR Mueller and B WoldScience 10 November 1989 246: 780-786 [DOI: 10.1126/science.2814500] (in Articles)
......muscle specific enhancer by ligation mediated PCR PR Mueller B Wold Division of Biology...newly developed polymerase chain reaction (PCR) footprinting procedure. This ligation mediated, single-sided PCR technique permits the exponential amplification......
Polymerase Chain Reaction: Promega Wins Round in Fight Over Taq
Marcia BarinagaScience 23 August 1996 273: 1039-0 [DOI: 10.1126/science.273.5278.1039a] (in News & Comment)
......legislation & jurisprudence Patents Polymerase Chain Reaction Taq Polymerase POLYMERASE CHAIN REACTION Promega Wins Round in Fight Over Taq...the popular technique known as the polymerase chain reaction (PCR)-took a new turn in a San Francisco......
Summary » PDF »
Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction
P Liang and AB PardeeScience 14 August 1992 257: 967-971 [DOI: 10.1126/science.1354393] (in Articles)
......messenger RNA by means of the polymerase chain reaction P Liang AB Pardee...mRNAs) by means of the polymerase chain reaction. The key element is to...messenger RNA by means of the polymerase chain reaction. Effective methods are......
Abstract » References » PDF »
Recent advances in the polymerase chain reaction
HA Erlich, D Gelfand, and JJ SninskyScience 21 June 1991 252: 1643-1651 [DOI: 10.1126/science.2047872] (in Articles)
......Recent advances in the polymerase chain reaction HA Erlich D Gelfand...Emeryville, CA 94608. The polymerase chain reaction (PCR) has dramatically...Recent advances in the polymerase chain reaction. The polymerase chain......
Abstract » References » PDF »
Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain
EY Loh, JF Elliott, S Cwirla, LL Lanier, and MM DavisScience 13 January 1989 243: 217-220 [DOI: 10.1126/science.2463672] (in Articles)
...articles Polymerase chain reaction with single-sided specificity...CA 94305-5402. In the polymerase chain reaction (PCR), two specific oligonucleotide...novel technique, anchored polymerase chain reaction (A-PCR), was devised that......
Abstract » References » PDF »
Variants of PCR (2)
PCR Serial and It's Related Protocols
PCR (General Procedure)
PCR Primer Design Tools
RT-PCR
Real time PCR
More PCR Protocols Online
Video and Animation of PCR
Mouse Genotyping by PCR
PCR Based Molecular Cloning
PCR Primer Design and Reaction Optimisation
10 Things That Can Kill Your PCR
PCR Troubleshootings
What's Polymerase Chain Reaction (PCR)? ...Principal,Procedure,and more...
The polymerase chain reaction (PCR) is a biochemistry and molecular biology technique[1] forexponentially amplifying a fragment of DNA, via enzymatic replication, without using a living organism (such as E. coli or yeast). PCR can be used for amplification of a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations.
Developed in 1983 by Kary Mullis, PCR is now a common technique used in medical and biological research labs for a variety of tasks, such as the sequencing of genes and the diagnosis ofhereditary diseases, the identification of genetic fingerprints (used in forensics and paternity testing), the detection and diagnosis of infectious diseases, and the creation of transgenic organisms. Mullis won the Nobel Prize for his work on PCR.
See: Wikipedia..
FRQs for PCR and other experiments on molecular biology
17. What is PCR? 18. What are some good references for PCR?19. How should I select a set of primers to use for PCR?20. What kinds of programs are available for designing PCR primers?21. What is "Hot-start" PCR? 22. What is AP-PCR or RAPD PCR?23. What is "Touchdown" PCR? 24. Is there a simple method to sequence lambda, M13, or plasmid clones using PCR?
34. Should we break up the methods-reagnts group into subsets with one beingexclusively on the polymerase chain reaction (PCR)?
In situ PCR: protocols and applications
Polymerase Chain Reaction (PCR) Animation
More PCR Protocols Online
PCR (General Procedure)
PCR Primer Design Tools
RT-PCR
Real time PCR
More PCR Protocols Online
Video and Animation of PCR
Mouse Genotyping by PCR
PCR Based Molecular Cloning
PCR Primer Design and Reaction Optimisation
10 Things That Can Kill Your PCR
PCR Troubleshootings
What's Polymerase Chain Reaction (PCR)? ...Principal,Procedure,and more...
The polymerase chain reaction (PCR) is a biochemistry and molecular biology technique[1] forexponentially amplifying a fragment of DNA, via enzymatic replication, without using a living organism (such as E. coli or yeast). PCR can be used for amplification of a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations.
Developed in 1983 by Kary Mullis, PCR is now a common technique used in medical and biological research labs for a variety of tasks, such as the sequencing of genes and the diagnosis ofhereditary diseases, the identification of genetic fingerprints (used in forensics and paternity testing), the detection and diagnosis of infectious diseases, and the creation of transgenic organisms. Mullis won the Nobel Prize for his work on PCR.
See: Wikipedia..
FRQs for PCR and other experiments on molecular biology
17. What is PCR? 18. What are some good references for PCR?19. How should I select a set of primers to use for PCR?20. What kinds of programs are available for designing PCR primers?21. What is "Hot-start" PCR? 22. What is AP-PCR or RAPD PCR?23. What is "Touchdown" PCR? 24. Is there a simple method to sequence lambda, M13, or plasmid clones using PCR?
34. Should we break up the methods-reagnts group into subsets with one beingexclusively on the polymerase chain reaction (PCR)?
In situ PCR: protocols and applications
Polymerase Chain Reaction (PCR) Animation
More PCR Protocols Online
PCR SSCP
Protocol: Mutation Detection by SSCP PCR.
A protocol for mutation detection by single-strand conformational polymorphism (SSCP) by PCR from the neurogenetics laboratory in the Neurological Sciences ...www.ohsu.edu/nsi/faculty/reddyh/lab/protsscp.html
step by step sscp
Step by Step SSCP. Travis Glenn. Laboratory of Molecular Systematics. Smithsonian Institution. Washington, DC 20560. phone: 301-238-3444. fax: 301-238-3059 ...www.uga.edu/srel/DNA_Lab/SSCP'96V2.rtf
Springer Protocols: Abstract: Multiple Fluorescence-Based PCR-SSCP analysis with primer, post- and internal labeling.
Springer Protocols is the largest subscription-based electronic database of reproducible laboratory protocols in the Life and Biomedical Sciences.www.springerprotocols.com/Abstract/doi/10.1385/0-89603-499-2:51
PCR-SSCP: a practical approach (detailed SSCP protocols)
The multiplexed PCR-SSCP analysis described here (Protocol 5) is essentially a two step procedure, each strand of the target DNA sequence is labelled as it ...europium.csc.mrc.ac.uk/WebPages/Database/Methods/pcrpract.htm
Optimization of Nonisotopic PCR–Single-Strand Conformation Polymorphism.
The protocol used for the GCK gene allowed us to establish a successful strategy for the development of PCR-SSCP on other genes such as BRCA1 (breast cancer ...www.clinchem.org/cgi/content/full/43/11/2190
Genomic Variation Laboratory – SSCP Protocol
Feb 27, 2003 ... SSCP Protocol. MDE gel (BioWhittaker Molecular Applications) final ... 3) For single strands, mix 2 ul PCR product for each sample with 10 ...http://genome-lab.ucdavis.edu/Protocols/SSCP%20Protocols.htm
Evaluating Duplicate Gene Expression using RT-PCR/SSCP Analysis (Wendel Lab.)
The subsequent PCR reaction follows the protocol you have predetermined to work best .... 1995, Identification of DNA polymorphism by asymmetric-PCR SSCP. ...
http://www.eeob.iastate.edu/faculty/WendelJ/rt-pcr_sscp.htm
Sensitive detection of p53 gene mutations by a 'mutant enriched PCR SSCP technique.
In the past, the existing PCR-SSCP technique as established by Orita et al. ... Figure 1 gives a schematic view of the protocol applied whereby the ...nar.oxfordjournals.org/cgi/content/full/26/5/1356
Single-strand conformational polymorphism.
cantly to its utility. SSCP PROTOCOL. The following is an example of an SSCP protocol that we used for detection. $138 PCR Methods and Applications ...www.genome.org/cgi/reprint/4/3/S137.pdf?ck=nck
A protocol for mutation detection by single-strand conformational polymorphism (SSCP) by PCR from the neurogenetics laboratory in the Neurological Sciences ...www.ohsu.edu/nsi/faculty/reddyh/lab/protsscp.html
step by step sscp
Step by Step SSCP. Travis Glenn. Laboratory of Molecular Systematics. Smithsonian Institution. Washington, DC 20560. phone: 301-238-3444. fax: 301-238-3059 ...www.uga.edu/srel/DNA_Lab/SSCP'96V2.rtf
Springer Protocols: Abstract: Multiple Fluorescence-Based PCR-SSCP analysis with primer, post- and internal labeling.
Springer Protocols is the largest subscription-based electronic database of reproducible laboratory protocols in the Life and Biomedical Sciences.www.springerprotocols.com/Abstract/doi/10.1385/0-89603-499-2:51
PCR-SSCP: a practical approach (detailed SSCP protocols)
The multiplexed PCR-SSCP analysis described here (Protocol 5) is essentially a two step procedure, each strand of the target DNA sequence is labelled as it ...europium.csc.mrc.ac.uk/WebPages/Database/Methods/pcrpract.htm
Optimization of Nonisotopic PCR–Single-Strand Conformation Polymorphism.
The protocol used for the GCK gene allowed us to establish a successful strategy for the development of PCR-SSCP on other genes such as BRCA1 (breast cancer ...www.clinchem.org/cgi/content/full/43/11/2190
Genomic Variation Laboratory – SSCP Protocol
Feb 27, 2003 ... SSCP Protocol. MDE gel (BioWhittaker Molecular Applications) final ... 3) For single strands, mix 2 ul PCR product for each sample with 10 ...http://genome-lab.ucdavis.edu/Protocols/SSCP%20Protocols.htm
Evaluating Duplicate Gene Expression using RT-PCR/SSCP Analysis (Wendel Lab.)
The subsequent PCR reaction follows the protocol you have predetermined to work best .... 1995, Identification of DNA polymorphism by asymmetric-PCR SSCP. ...
http://www.eeob.iastate.edu/faculty/WendelJ/rt-pcr_sscp.htm
Sensitive detection of p53 gene mutations by a 'mutant enriched PCR SSCP technique.
In the past, the existing PCR-SSCP technique as established by Orita et al. ... Figure 1 gives a schematic view of the protocol applied whereby the ...nar.oxfordjournals.org/cgi/content/full/26/5/1356
Single-strand conformational polymorphism.
cantly to its utility. SSCP PROTOCOL. The following is an example of an SSCP protocol that we used for detection. $138 PCR Methods and Applications ...www.genome.org/cgi/reprint/4/3/S137.pdf?ck=nck
PCR RFLP
Restriction fragment length polymorphism (From Wikipedia, the free encyclopedia):
A Restriction Fragment Length Polymorphism (or RFLP, often pronounced as "rif-lip") is a variation in the DNA sequence of a genome which can be detected by a laboratory technique known as gel electrophoresis. Analysis of RFLP variation was an important tool in genome mapping, localization of genetic disease genes, determination of risk for a disease, genetic fingerprinting, and paternity testing.
Contents
1 Analysis technique
2 Examples
3 Applications
4 Alternatives
5 References
5.1 External links
PCR-RFLP Method
18-Oct-02.www.ihwg.org/components/cytokine/mon8Lin.htm
Examples of PCR-RFLP for Nematode Diagnostics
Examples of Restriction Fragment Length Polymorphism (RFLP)electrophoresis slabs for different nematodes, from University of Nebrasca.nematode.unl.edu/its_id/EXAMPLES/index.htm
Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences
Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences.www.kfunigraz.ac.at/~binder/thesis/node64.html
RFLP/PCR Polymorphism Query Form
Search for RFLP and PCR based polymorphisms by strain, locus symbol, or map position. To search for SNPS, use the SNP Query Form. ...www.informatics.jax.org/searches/polymorphism_form.shtml
PCR, RFLP and Gene Therapy
Lecture 24:Genetic Engineering: PCR, RFLP Analysis & Gene Therapy. The Polymerase Chain Reaction (PCR) Can Make Millions of Copies of DNA in a Short Time ...http://members.aol.com/BearFlag45/Biology1A/LectureNotes/lec24.html
Handbook for DNA isolation, RAPD-PCR and PCR-RFLP
General protocol. We use agarose gels for checking the quality of DNA isolates, PCR products, and PCR-RFLP products, and for scoring RAPD products. ...www.toyen.uio.no/botanisk/brochmann/handbook.htm
PCR-RFLP PROTOCOL FOR ALLELES A AND B PCR ...BOVINE KAPPA-CASEIN PCR-RFLP PROTOCOL. FOR ALLELES A AND B. Laboratory of J.F. Medrano. Department of Animal Science. University of California. ...animalscience.ucdavis.edu/laboratory/
A Restriction Fragment Length Polymorphism (or RFLP, often pronounced as "rif-lip") is a variation in the DNA sequence of a genome which can be detected by a laboratory technique known as gel electrophoresis. Analysis of RFLP variation was an important tool in genome mapping, localization of genetic disease genes, determination of risk for a disease, genetic fingerprinting, and paternity testing.
Contents
1 Analysis technique
2 Examples
3 Applications
4 Alternatives
5 References
5.1 External links
PCR-RFLP Method
18-Oct-02.www.ihwg.org/components/cytokine/mon8Lin.htm
Examples of PCR-RFLP for Nematode Diagnostics
Examples of Restriction Fragment Length Polymorphism (RFLP)electrophoresis slabs for different nematodes, from University of Nebrasca.nematode.unl.edu/its_id/EXAMPLES/index.htm
Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences
Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences.www.kfunigraz.ac.at/~binder/thesis/node64.html
RFLP/PCR Polymorphism Query Form
Search for RFLP and PCR based polymorphisms by strain, locus symbol, or map position. To search for SNPS, use the SNP Query Form. ...www.informatics.jax.org/searches/polymorphism_form.shtml
PCR, RFLP and Gene Therapy
Lecture 24:Genetic Engineering: PCR, RFLP Analysis & Gene Therapy. The Polymerase Chain Reaction (PCR) Can Make Millions of Copies of DNA in a Short Time ...http://members.aol.com/BearFlag45/Biology1A/LectureNotes/lec24.html
Handbook for DNA isolation, RAPD-PCR and PCR-RFLP
General protocol. We use agarose gels for checking the quality of DNA isolates, PCR products, and PCR-RFLP products, and for scoring RAPD products. ...www.toyen.uio.no/botanisk/brochmann/handbook.htm
PCR-RFLP PROTOCOL FOR ALLELES A AND B PCR ...BOVINE KAPPA-CASEIN PCR-RFLP PROTOCOL. FOR ALLELES A AND B. Laboratory of J.F. Medrano. Department of Animal Science. University of California. ...animalscience.ucdavis.edu/laboratory/
Variants of PCR (1)
From Wikipedia, the free encyclopedia
This page assumes familiarity with the terms and components used in the Polymerase Chain Reaction (PCR) process.
The versatility of PCR has led to a large number of variants:
Contents
Basic modifications
Pretreatments and extensions
Buffer and temperature modifications
Primer modifications
Polymerase modifications
Mechanism modifications
Isothermal amplification methods
Additional reading
References
Basic modifications
Often only a small modification needs to be made to the 'standard' PCR protocol to achieve a desired goal:
One of the first adjustments made to PCR was the amplification of more than one target in a single tube. Multiplex-PCR can involve up to a dozen pairs of primers acting independently. This modification might be used simply to avoid having to prepare many individual reactions, or could allow the simultaneous analysis of multiple targets in a sample that has only a single copy of a genome. In testing for genetic disease mutations, six or more amplifications might be combined. In the standard protocol for DNA Fingerprinting, the 13 targets assayed are often amplified in groups of 3 or 4. Multiplex Ligation-dependent Probe Amplification (or MLPA) permits multiple targets to be amplified using only a single pair or primers, avoiding the resolution limitations of multiplex PCR.
VNTR PCR involves few modifications to the basic PCR process, but instead targets areas of the genome that exhibit length variation. The analysis of the genotypes of the sample usually involves simple sizing of the amplification products by gel electrophoresis. Analysis of smaller VNTR segments known as Short Tandem Repeats (or STRs) is the basis for DNA Fingerprinting databases such as CODIS.
Asymmetric PCR is used to preferentially amplify one strand of the target DNA. It finds use in some types of sequencing and hybridization probing, where having only one of the two complementary strands of the product is advantageous. PCR is carried out as usual, but with a limiting amount of one of the primers. When it becomes depleted, continued replication leads to an arithmetic increase in extension of the other primer[1]. A recent modification on this process, known as Linear-After-The-Exponential-PCR (or LATE-PCR), uses a limiting primer with a higher melting temperature Melting temperature (or Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction[2]. (Also see Overlap-extension PCR).
Some modifications are needed to perform long PCR. The original Klenow-based PCR process had trouble making a product larger than about 400 bp. However, early characterization of Taq polymerase showed that it could amplify targets up to several thousand bp long[3]. Since then, modified protocols have allowed targets of over 50,000 bp to be amplified[4].
Nested PCR, another early modification, can be used to increase the specificity of DNA amplification. Two sets of primers are used in two successive reactions. In the first, one pair of primers is used to generate DNA products, which may also contain products amplified from non-target areas. The products from the first PCR are then used to start a second, using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set. The specificity of all of the primers is combined, usually leading to a single product. Nested PCR is often more successful in specifically amplifying long DNA products than conventional PCR, but it requires more detailed knowledge of the sequence of the target.
Quantitative PCR (or Q-PCR) is used to measure the specific amount of target DNA (or RNA) in a sample. The normal PCR process is performed in a way that is largely qualitative - the amount of final product is only slightly proportional to the initial amount of target. By carefully running the amplification only within the phase of true exponential increase (avoiding the later 'plateau' phase), the amount of product is more proportional to the initial amount of target. Thermal cyclers have been developed which can monitor the amount of product during the amplification, allowing quantitation of samples containing a wide range of target copies. A method currently used is Quantitative Real-Time PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product as the amplification progresses. It is often confusingly referred to as RT-PCR, the same acronym used for PCR combined with Reverse Transcriptase (see below), which itself might be used in conjunction with Q-PCR. More appropriate acronyms are QRT-PCR or RTQ-PCR.
Hot-start PCR is a technique that modifies the way that a PCR mixture is initially heated. During this step the polymerase is active, but the target has not yet been denatured and the primers may be able to bind to non-specific locations (or even to each other). The technique can be performed manually by heating the reaction components to the melting temperature (e.g. 95°C) before adding the polymerase[5]. Alternatively, specialized systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody, or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. 'Hot-start/cold-finish PCR' is achieved with new hybrid polymerases that are inactive at ambient temperature and are only activated at elevated temperatures.
Another simple modification can also decrease non-specific amplification. In Touchdown PCR, the temperature used to anneal the primers is gradually decreased in later cycles. The annealing temperature in the early cycles is usually 3-5°C above the standard Tm of the primers used, while in the later cycles it is a similar amount below the Tm. The initial higher annealing temperature leads to greater specificity for primer binding, while the lower temperatures permit more efficient amplification to the end of the reaction[6].
Other common modifications to PCR allow it to amplify low copy targets. The original report on Taq polymerase[3] showed how the use of up to 60 cycles could amplify targets diluted to just one copy per reaction tube. A later report[7] showed how multiple genetic loci could be amplified and analyzed from a single sperm. Modified protocols[8] have allowed the identification of just one copy of the HIV genome within the DNA of up to 70,000 host cells.
Assembly PCR (also known as Polymerase Cycling Assembly or PCA) is the artificial synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotide building blocks alternate between sense and antisense directions, and the overlaps determine the order of oligonucleotides, thereby selectively producing the final long DNA product[9].
In Colony PCR, bacterial colonies are rapidly screened by PCR for correct DNA vector constructs. Colonies are sampled with a sterile toothpick and dabbed into a master mix. To free the DNA for amplification, PCR is either started with an extended time at 95°C (when standard polymerase is used), or with a shortened denaturation step at 100°C and special chimeric DNA polymerase[10]. Colonies from the master mix that shows the desired product are then tested individually.
The Digital polymerase chain reaction simultaneously amplifies thousands of samples, each in a separate droplet within an emulsion.
Pretreatments and extensions
The basic PCR process can sometimes precede or follow another technique:
RT-PCR (or Reverse Transcription PCR) is a common method used to amplify, isolate, or identify a known sequence from a cell's or tissue's RNA. PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. The two reactions are compatible enough that they can be run in the same mixture tube, with the initial heating step of PCR being used to inactivate the transcriptase[3]. Also, the Tth polymerase described below exhibits RT activity, and can carry out the entire combined reaction. RT-PCR is widely used in expression profiling, which determines the expression of a gene or identifies the sequence of an RNA transcript (including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene). The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method named RACE-PCR, short for Rapid Amplification of cDNA Ends. (Note that the acronym RT-PCR has more recently been applied to Real-Time PCR, a version of Quantitative PCR described above.)
Since PCR is based on components of DNA replication, it is not surprising that it can easily be combined[1] with DNA sequencing. In its simplest form, the products of an 'asymmetric PCR' (above) are diluted into a new reaction containing sequencing components, which are then extended by Taq polymerase.
Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' that are first ligated to fragments of the target DNA. PCR primers are then chosen from the linker sequences, and used to amplify the unknown target fragments. It has been used for DNA sequencing, genome walking, and DNA footprinting[11]. A related technique, Amplified fragment length polymorphism, looks at fragments of a genome that differ in length.
Methylation-specific PCR (or MSP) was developed to study patterns of methylation at CpG islands in genomic DNA[12]. Target DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two amplifications are then carried out on the modified DNA, using primer sets that distinguish between the modified and unmodified templates. One primer set recognizes DNA with cytosines to amplify the previously methylated DNA, and the other set recognizes DNA with uracil or thymine to amplify unmethylated target. MSP using Q-PCR can also be performed to obtain quantitative information about methylation.
Buffer and temperature modifications
Adjustments to the 'small' components in PCR can sometimes be useful:
The divalent magnesium ion (Mg++) is crucial to the activity of the polymerase used in PCR. Since many of the other components used in an amplification will also bind Mg++, it's exact concentration available to the enzyme is difficult to control. In general, lower concentrations will increase replication fidelity, while higher concentrations will introduce more mutations (either of which may be desired).
The use in PCR of modified dNTPs can help to control 'carryover' contamination. The PCR process can be carried out using dUTP, an analog of the normal dTTP. Later amplifications are then treated with an enzyme that destroys DNA containing the analog, but leaving the normal target DNA unmodified[13]. Thus, targets that represent contamination from earlier amplifications are selectively destroyed.
A wide variety of other chemicals can be added to PCR, for a variety of effects. Mild denaturants (such as DMSO) can increase amplification specificity by destabilizing non-specific primer binding. Certain chemicals (such as glycerol) can act as stabilizers for the activity of the polymerase during amplification. Detergents (such as Triton X-100) can prevent having the polymerase stick to itself, or to the walls of the reaction tube.
The temperature changes carried out by the thermal cycler will also affect amplification. A particular set of primers are usually tested using different annealing temperatures to determine their optimum. The time given to the polymerase to fully copy the templates may need to be adjusted, depending on their lengths. Longer extension times can also lead to higher yields after the reaction has entered the 'plateau' phase. When amplifying low-copy targets, the total number of cycles performed must be increased.
The polymerases that perform replication during PCR sometime incorporate incorrect bases. This is of no consequence to most assays that test the bulk of the amplified product - the errors are scattered within the product at random, and aren't seen by the assay. However, it is best to perform high-fidelity PCR when the products are individually cloned (for sequencing or expression). A different DNA polymerase (such as Pfu, with a proofreading activity missing in Taq) might be used, and the Mg++ and dNTP concentrations might be adjusted to maximize the number of products that exactly match the original target DNA. Some researchers choose to do the opposite, purposefully running PCR under low-fidelity conditions to produce a spectrum of mutations in the amplified product.
(For additional details, see the auxiliary article PCR optimization.)
Primer modifications
Adjustments to the synthetic oligonucleotides used as primers in PCR are a rich source of modification:
Normally PCR primers are chosen from an invariant part of the genome, and might be used to amplify a polymorphic area between them. In Allele-specific PCR the opposite is done. At least one of the primers is chosen from a polymorphic area, with the mutations located at (or near) its 3'-end. Under stringent conditions, a mismatched primer will not initiate replication, whereas a matched primer will. The appearance of an amplification product therefore indicates the genotype. (For more information, see SNP genotyping.)
InterSequence-Specific PCR (or ISSR-PCR) is method for DNA fingerprinting that uses primers selected from segments repeated throughout a genome to produce a unique fingerprint of amplified product lengths[14]. The use of primers from a commonly repeated segment is called Alu-PCR, and can help amplify sequences adjacent (or between) these repeats.
Primers can also be designed to be 'degenerate' - able to initiate replication from a large number of target locations. Whole genome amplification (or WGA) is a group of procedures that allow amplification to occur at many locations in an unknown genome, and which may only be available in small quantities. Other techniques use degenerate primers that are synthesized using multiple nucleotides at particular positions (the polymerase 'chooses' the correctly matched primers). Also, the primers can be synthesized with the nucleoside analog inosine, which hybridizes to three of the four normal bases. A similar technique can force PCR to perform Site-directed mutagenesis. (also see Overlap extension polymerase chain reaction)
Normally the primers used in PCR are designed to be fully complementary to the target. However, the polymerase is tolerant to mis-matches away from the 3' end. Tailed-primers include non-complementary sequences at their 5' ends. A common procedure is the use of linker-primers, which ultimately place restriction sites at the ends of the PCR products, facilitating their later insertion into cloning vectors.
An extension of the 'colony-PCR' method (above), is the use of vector primers. Target DNA fragments (or cDNA) are first inserted into a cloning vector, and a single set of primers are designed for the areas of the vector flanking the insertion site. Amplification occurs for whatever DNA has been inserted[3].
PCR can easily be modified to produce a labeled product for subsequent use as a hybridization probe. One or both primers might be used in PCR with a radioactive or fluorescent label already attached, or labels might be added after amplification. These labeling methods can be combined with 'asymmetric-PCR' (above) to produce effective hybridization probes.
Polymerase modifications
There are many choices for the all-important DNA polymerase used in PCR:
The Klenow fragment, derived from the original DNA Polymerase I from E. coli, was the first enzyme used to demonstrate PCR. It is inactivated in the denaturation step of PCR, and had to be replenished during each cycle.
The bacteriophage T4 DNA polymerase was also tested shortly after the first reports of PCR. It has a higher fidelity of replication than the Klenow fragment. Since it is also destroyed by heat, it has seen little use since the development of thermostable polymerases.
The DNA polymerase from Thermus aquaticus (or Taq), was the first thermostable polymerase used in PCR[3], and is still the one most commonly used. The enzyme can be isolated from its 'native' bacterial source, or from a cloned gene expressed in E. coli.
The Stoffel fragment is produced from a truncated gene for Taq polymerase, expressed in E. coli. It is missing the 'forward' nuclease activity, and may be able to amplify longer targets than the native enzyme.
The Faststart polymerase is a variant of Taq polymerase that only becomes active after the first denaturation step of PCR, thereby avoiding problems during the first cycle. (see Hot-start PCR above)
A thermostable polymerase has also been isolated from the archeozoic organism Pyrococcus furiosus. Unlike Taq polymerase, Pfu DNA polymerase includes a 'proofreading' activity, leading to about a 5-fold decrease in the error rate of replication[15]. Since these errors accumulate during every cycle of PCR, Pfu is the preferred polymerase when products are to be individually cloned for sequencing or expression.
An extremely thermostable DNA polymerase has been isolated from Thermococcus litoralis, and is marketed as Vent polymerase.
Another thermostable polymerase has been isolated from Thermus thermophilus, and is known as Tth polymerase. In the presence of Mn++ ions, it exhibits a reverse transcriptase activity, allowing PCR amplification to be initiated by RNA targets.
But not Bst polymerase, isolated from the thermophilic bacterium Bacillus stearothermophilus. This was an early candidate to be tested for PCR. It was later found to be unsuitable for continued amplification - it is irreversibly inactivated during the denaturation step. This highlights the point that a good polymerase for PCR should both be active at a higher temperature (for specificity), and should also be able to survive the near-boiling temperatures of the PCR process.
Mechanism modifications
Sometimes even the basic mechanism of PCR can be modified:
Unlike normal PCR, Inverse PCR allows amplification and sequencing of DNA that surrounds a known sequence. It involves initially subjecting the target DNA to a series of restriction enzyme digestions, and then circularizing the resulting fragments by self ligation. Primers are designed to be extended outward from the known segment, resulting in amplification of the rest of the circle. This is especially useful in identifying sequences to either side of various genomic inserts[16].
Similarly, Thermal Asymmetric InterLaced PCR (or TAIL-PCR) is used to isolate unknown sequences flanking a known area of the genome. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures. A 'degenerate' primer is used to amplify in the other direction from the unknown sequence[17].
Isothermal amplification methods
Some amplification protocols have been developed that only remotely resemble PCR:
Helicase-dependent amplification is a technique that is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension steps. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation[18].
PAN-AC also uses isothermal conditions for amplification, and may be used to analyze living cells[19][20].
Additional reading
PCR Applications Manual (from Roche Diagnostics).]
References
1. ^ a b Innis MA, Myambo KB, Gelfand DH, Brow MA. (1988). "DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA". Proc Natl Acad Sci USA 85: 9436-4940. PMID 3200828.
2. ^ Pierce KE and Wangh LJ (2007). "Linear-after-the-exponential polymerase chain reaction and allied technologies Real-time detection strategies for rapid, reliable diagnosis from single cells". Methods Mol Med. 132: 65-85. PMID 17876077.
3. ^ a b c d e Saiki et al. "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science vol. 239 pp. 487-91 (1988).
4. ^ Cheng S, Fockler C, Barnes WM, Higuchi R. "Effective amplification of long targets from cloned inserts and human genomic DNA." Proc Natl Acad Sci vol. 91(12) pp. 5695-9 (1994).
5. ^ Q. Chou, M. Russell, D.E. Birch, J. Raymond and W. Bloch (1992). "Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications". Nucleic Acids Research 20: 1717-1723.
6. ^ Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991). "'Touchdown' PCR to circumvent spurious priming during gene amplification.". Nucl Acids Res 19: 4008.
7. ^ Boehnke M et al. "Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm." Am J Hum Genet vol. 45(1) pp. 21-32 (1989).
8. ^ Kwok S et al. "Identification of HIV sequences by using in vitro enzymatic amplification and oligomer cleavage detection." J. Virol. vol. 61(5) pp. 1690-4 (1987).
9. ^ Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides". Gene 164: 49-53. PMID 7590320.
10. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes", in Kieleczawa J: DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett, pp. 241-257. ISBN 0-7637338-3-0.
11. ^ Mueller PR, Wold B (1988). "In vivo footprinting of a muscle specific enhancer by ligation mediated PCR". Science 246: 780-786. PMID 2814500.
12. ^ Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB (1996). "Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands". Proc Natl Acad Sci U S A 93 (13): 9821-9826. PMID 8790415.
13. ^ Longo MC, Berninger MS, Hartley JL "Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions." Gene vol. 93(1) pp. 125-8 (1990).
14. ^ E. Zietkiewicz, A. Rafalski, and D. Labuda (1994). "Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification". Genomics 20 (2): 176-83.
15. ^ Cline J,Braman JC, Hogrefe HH "PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases." Nucleic Acids Research vol. 24(18) pp. 3546-51 (1996).
16. ^ Ochman H, Gerber AS, Hartl DL (1988). "Genetic applications of an inverse polymerase chain reaction". Genetics 120: 621-623. PMID 2852134.
17. ^ Y.G. Liu and R. F. Whittier (1995). "Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking". Genomics 25 (3): 674-81.
18. ^ Myriam Vincent, Yan Xu and Huimin Kong (2004). "Helicase-dependent isothermal DNA amplification". EMBO reports 5 (8): 795–800.
19. ^ David, F.Turlotte, E., (1998). "An Isothermal Amplification Method". C.R.Acad. Sci Paris, Life Science 321 (1): 909-914.
20. ^ Fabrice David (September-October 2002). Utiliser les propriétés topologiques de l’ADN: une nouvelle arme contre les agents pathogènes. Fusion.(in French)
Retrieved from "http://en.wikipedia.org/wiki/Variants_of_PCR"
This page assumes familiarity with the terms and components used in the Polymerase Chain Reaction (PCR) process.
The versatility of PCR has led to a large number of variants:
Contents
Basic modifications
Pretreatments and extensions
Buffer and temperature modifications
Primer modifications
Polymerase modifications
Mechanism modifications
Isothermal amplification methods
Additional reading
References
Basic modifications
Often only a small modification needs to be made to the 'standard' PCR protocol to achieve a desired goal:
One of the first adjustments made to PCR was the amplification of more than one target in a single tube. Multiplex-PCR can involve up to a dozen pairs of primers acting independently. This modification might be used simply to avoid having to prepare many individual reactions, or could allow the simultaneous analysis of multiple targets in a sample that has only a single copy of a genome. In testing for genetic disease mutations, six or more amplifications might be combined. In the standard protocol for DNA Fingerprinting, the 13 targets assayed are often amplified in groups of 3 or 4. Multiplex Ligation-dependent Probe Amplification (or MLPA) permits multiple targets to be amplified using only a single pair or primers, avoiding the resolution limitations of multiplex PCR.
VNTR PCR involves few modifications to the basic PCR process, but instead targets areas of the genome that exhibit length variation. The analysis of the genotypes of the sample usually involves simple sizing of the amplification products by gel electrophoresis. Analysis of smaller VNTR segments known as Short Tandem Repeats (or STRs) is the basis for DNA Fingerprinting databases such as CODIS.
Asymmetric PCR is used to preferentially amplify one strand of the target DNA. It finds use in some types of sequencing and hybridization probing, where having only one of the two complementary strands of the product is advantageous. PCR is carried out as usual, but with a limiting amount of one of the primers. When it becomes depleted, continued replication leads to an arithmetic increase in extension of the other primer[1]. A recent modification on this process, known as Linear-After-The-Exponential-PCR (or LATE-PCR), uses a limiting primer with a higher melting temperature Melting temperature (or Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction[2]. (Also see Overlap-extension PCR).
Some modifications are needed to perform long PCR. The original Klenow-based PCR process had trouble making a product larger than about 400 bp. However, early characterization of Taq polymerase showed that it could amplify targets up to several thousand bp long[3]. Since then, modified protocols have allowed targets of over 50,000 bp to be amplified[4].
Nested PCR, another early modification, can be used to increase the specificity of DNA amplification. Two sets of primers are used in two successive reactions. In the first, one pair of primers is used to generate DNA products, which may also contain products amplified from non-target areas. The products from the first PCR are then used to start a second, using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set. The specificity of all of the primers is combined, usually leading to a single product. Nested PCR is often more successful in specifically amplifying long DNA products than conventional PCR, but it requires more detailed knowledge of the sequence of the target.
Quantitative PCR (or Q-PCR) is used to measure the specific amount of target DNA (or RNA) in a sample. The normal PCR process is performed in a way that is largely qualitative - the amount of final product is only slightly proportional to the initial amount of target. By carefully running the amplification only within the phase of true exponential increase (avoiding the later 'plateau' phase), the amount of product is more proportional to the initial amount of target. Thermal cyclers have been developed which can monitor the amount of product during the amplification, allowing quantitation of samples containing a wide range of target copies. A method currently used is Quantitative Real-Time PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product as the amplification progresses. It is often confusingly referred to as RT-PCR, the same acronym used for PCR combined with Reverse Transcriptase (see below), which itself might be used in conjunction with Q-PCR. More appropriate acronyms are QRT-PCR or RTQ-PCR.
Hot-start PCR is a technique that modifies the way that a PCR mixture is initially heated. During this step the polymerase is active, but the target has not yet been denatured and the primers may be able to bind to non-specific locations (or even to each other). The technique can be performed manually by heating the reaction components to the melting temperature (e.g. 95°C) before adding the polymerase[5]. Alternatively, specialized systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody, or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. 'Hot-start/cold-finish PCR' is achieved with new hybrid polymerases that are inactive at ambient temperature and are only activated at elevated temperatures.
Another simple modification can also decrease non-specific amplification. In Touchdown PCR, the temperature used to anneal the primers is gradually decreased in later cycles. The annealing temperature in the early cycles is usually 3-5°C above the standard Tm of the primers used, while in the later cycles it is a similar amount below the Tm. The initial higher annealing temperature leads to greater specificity for primer binding, while the lower temperatures permit more efficient amplification to the end of the reaction[6].
Other common modifications to PCR allow it to amplify low copy targets. The original report on Taq polymerase[3] showed how the use of up to 60 cycles could amplify targets diluted to just one copy per reaction tube. A later report[7] showed how multiple genetic loci could be amplified and analyzed from a single sperm. Modified protocols[8] have allowed the identification of just one copy of the HIV genome within the DNA of up to 70,000 host cells.
Assembly PCR (also known as Polymerase Cycling Assembly or PCA) is the artificial synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotide building blocks alternate between sense and antisense directions, and the overlaps determine the order of oligonucleotides, thereby selectively producing the final long DNA product[9].
In Colony PCR, bacterial colonies are rapidly screened by PCR for correct DNA vector constructs. Colonies are sampled with a sterile toothpick and dabbed into a master mix. To free the DNA for amplification, PCR is either started with an extended time at 95°C (when standard polymerase is used), or with a shortened denaturation step at 100°C and special chimeric DNA polymerase[10]. Colonies from the master mix that shows the desired product are then tested individually.
The Digital polymerase chain reaction simultaneously amplifies thousands of samples, each in a separate droplet within an emulsion.
Pretreatments and extensions
The basic PCR process can sometimes precede or follow another technique:
RT-PCR (or Reverse Transcription PCR) is a common method used to amplify, isolate, or identify a known sequence from a cell's or tissue's RNA. PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. The two reactions are compatible enough that they can be run in the same mixture tube, with the initial heating step of PCR being used to inactivate the transcriptase[3]. Also, the Tth polymerase described below exhibits RT activity, and can carry out the entire combined reaction. RT-PCR is widely used in expression profiling, which determines the expression of a gene or identifies the sequence of an RNA transcript (including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene). The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method named RACE-PCR, short for Rapid Amplification of cDNA Ends. (Note that the acronym RT-PCR has more recently been applied to Real-Time PCR, a version of Quantitative PCR described above.)
Since PCR is based on components of DNA replication, it is not surprising that it can easily be combined[1] with DNA sequencing. In its simplest form, the products of an 'asymmetric PCR' (above) are diluted into a new reaction containing sequencing components, which are then extended by Taq polymerase.
Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' that are first ligated to fragments of the target DNA. PCR primers are then chosen from the linker sequences, and used to amplify the unknown target fragments. It has been used for DNA sequencing, genome walking, and DNA footprinting[11]. A related technique, Amplified fragment length polymorphism, looks at fragments of a genome that differ in length.
Methylation-specific PCR (or MSP) was developed to study patterns of methylation at CpG islands in genomic DNA[12]. Target DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two amplifications are then carried out on the modified DNA, using primer sets that distinguish between the modified and unmodified templates. One primer set recognizes DNA with cytosines to amplify the previously methylated DNA, and the other set recognizes DNA with uracil or thymine to amplify unmethylated target. MSP using Q-PCR can also be performed to obtain quantitative information about methylation.
Buffer and temperature modifications
Adjustments to the 'small' components in PCR can sometimes be useful:
The divalent magnesium ion (Mg++) is crucial to the activity of the polymerase used in PCR. Since many of the other components used in an amplification will also bind Mg++, it's exact concentration available to the enzyme is difficult to control. In general, lower concentrations will increase replication fidelity, while higher concentrations will introduce more mutations (either of which may be desired).
The use in PCR of modified dNTPs can help to control 'carryover' contamination. The PCR process can be carried out using dUTP, an analog of the normal dTTP. Later amplifications are then treated with an enzyme that destroys DNA containing the analog, but leaving the normal target DNA unmodified[13]. Thus, targets that represent contamination from earlier amplifications are selectively destroyed.
A wide variety of other chemicals can be added to PCR, for a variety of effects. Mild denaturants (such as DMSO) can increase amplification specificity by destabilizing non-specific primer binding. Certain chemicals (such as glycerol) can act as stabilizers for the activity of the polymerase during amplification. Detergents (such as Triton X-100) can prevent having the polymerase stick to itself, or to the walls of the reaction tube.
The temperature changes carried out by the thermal cycler will also affect amplification. A particular set of primers are usually tested using different annealing temperatures to determine their optimum. The time given to the polymerase to fully copy the templates may need to be adjusted, depending on their lengths. Longer extension times can also lead to higher yields after the reaction has entered the 'plateau' phase. When amplifying low-copy targets, the total number of cycles performed must be increased.
The polymerases that perform replication during PCR sometime incorporate incorrect bases. This is of no consequence to most assays that test the bulk of the amplified product - the errors are scattered within the product at random, and aren't seen by the assay. However, it is best to perform high-fidelity PCR when the products are individually cloned (for sequencing or expression). A different DNA polymerase (such as Pfu, with a proofreading activity missing in Taq) might be used, and the Mg++ and dNTP concentrations might be adjusted to maximize the number of products that exactly match the original target DNA. Some researchers choose to do the opposite, purposefully running PCR under low-fidelity conditions to produce a spectrum of mutations in the amplified product.
(For additional details, see the auxiliary article PCR optimization.)
Primer modifications
Adjustments to the synthetic oligonucleotides used as primers in PCR are a rich source of modification:
Normally PCR primers are chosen from an invariant part of the genome, and might be used to amplify a polymorphic area between them. In Allele-specific PCR the opposite is done. At least one of the primers is chosen from a polymorphic area, with the mutations located at (or near) its 3'-end. Under stringent conditions, a mismatched primer will not initiate replication, whereas a matched primer will. The appearance of an amplification product therefore indicates the genotype. (For more information, see SNP genotyping.)
InterSequence-Specific PCR (or ISSR-PCR) is method for DNA fingerprinting that uses primers selected from segments repeated throughout a genome to produce a unique fingerprint of amplified product lengths[14]. The use of primers from a commonly repeated segment is called Alu-PCR, and can help amplify sequences adjacent (or between) these repeats.
Primers can also be designed to be 'degenerate' - able to initiate replication from a large number of target locations. Whole genome amplification (or WGA) is a group of procedures that allow amplification to occur at many locations in an unknown genome, and which may only be available in small quantities. Other techniques use degenerate primers that are synthesized using multiple nucleotides at particular positions (the polymerase 'chooses' the correctly matched primers). Also, the primers can be synthesized with the nucleoside analog inosine, which hybridizes to three of the four normal bases. A similar technique can force PCR to perform Site-directed mutagenesis. (also see Overlap extension polymerase chain reaction)
Normally the primers used in PCR are designed to be fully complementary to the target. However, the polymerase is tolerant to mis-matches away from the 3' end. Tailed-primers include non-complementary sequences at their 5' ends. A common procedure is the use of linker-primers, which ultimately place restriction sites at the ends of the PCR products, facilitating their later insertion into cloning vectors.
An extension of the 'colony-PCR' method (above), is the use of vector primers. Target DNA fragments (or cDNA) are first inserted into a cloning vector, and a single set of primers are designed for the areas of the vector flanking the insertion site. Amplification occurs for whatever DNA has been inserted[3].
PCR can easily be modified to produce a labeled product for subsequent use as a hybridization probe. One or both primers might be used in PCR with a radioactive or fluorescent label already attached, or labels might be added after amplification. These labeling methods can be combined with 'asymmetric-PCR' (above) to produce effective hybridization probes.
Polymerase modifications
There are many choices for the all-important DNA polymerase used in PCR:
The Klenow fragment, derived from the original DNA Polymerase I from E. coli, was the first enzyme used to demonstrate PCR. It is inactivated in the denaturation step of PCR, and had to be replenished during each cycle.
The bacteriophage T4 DNA polymerase was also tested shortly after the first reports of PCR. It has a higher fidelity of replication than the Klenow fragment. Since it is also destroyed by heat, it has seen little use since the development of thermostable polymerases.
The DNA polymerase from Thermus aquaticus (or Taq), was the first thermostable polymerase used in PCR[3], and is still the one most commonly used. The enzyme can be isolated from its 'native' bacterial source, or from a cloned gene expressed in E. coli.
The Stoffel fragment is produced from a truncated gene for Taq polymerase, expressed in E. coli. It is missing the 'forward' nuclease activity, and may be able to amplify longer targets than the native enzyme.
The Faststart polymerase is a variant of Taq polymerase that only becomes active after the first denaturation step of PCR, thereby avoiding problems during the first cycle. (see Hot-start PCR above)
A thermostable polymerase has also been isolated from the archeozoic organism Pyrococcus furiosus. Unlike Taq polymerase, Pfu DNA polymerase includes a 'proofreading' activity, leading to about a 5-fold decrease in the error rate of replication[15]. Since these errors accumulate during every cycle of PCR, Pfu is the preferred polymerase when products are to be individually cloned for sequencing or expression.
An extremely thermostable DNA polymerase has been isolated from Thermococcus litoralis, and is marketed as Vent polymerase.
Another thermostable polymerase has been isolated from Thermus thermophilus, and is known as Tth polymerase. In the presence of Mn++ ions, it exhibits a reverse transcriptase activity, allowing PCR amplification to be initiated by RNA targets.
But not Bst polymerase, isolated from the thermophilic bacterium Bacillus stearothermophilus. This was an early candidate to be tested for PCR. It was later found to be unsuitable for continued amplification - it is irreversibly inactivated during the denaturation step. This highlights the point that a good polymerase for PCR should both be active at a higher temperature (for specificity), and should also be able to survive the near-boiling temperatures of the PCR process.
Mechanism modifications
Sometimes even the basic mechanism of PCR can be modified:
Unlike normal PCR, Inverse PCR allows amplification and sequencing of DNA that surrounds a known sequence. It involves initially subjecting the target DNA to a series of restriction enzyme digestions, and then circularizing the resulting fragments by self ligation. Primers are designed to be extended outward from the known segment, resulting in amplification of the rest of the circle. This is especially useful in identifying sequences to either side of various genomic inserts[16].
Similarly, Thermal Asymmetric InterLaced PCR (or TAIL-PCR) is used to isolate unknown sequences flanking a known area of the genome. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures. A 'degenerate' primer is used to amplify in the other direction from the unknown sequence[17].
Isothermal amplification methods
Some amplification protocols have been developed that only remotely resemble PCR:
Helicase-dependent amplification is a technique that is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension steps. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation[18].
PAN-AC also uses isothermal conditions for amplification, and may be used to analyze living cells[19][20].
Additional reading
PCR Applications Manual (from Roche Diagnostics).]
References
1. ^ a b Innis MA, Myambo KB, Gelfand DH, Brow MA. (1988). "DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA". Proc Natl Acad Sci USA 85: 9436-4940. PMID 3200828.
2. ^ Pierce KE and Wangh LJ (2007). "Linear-after-the-exponential polymerase chain reaction and allied technologies Real-time detection strategies for rapid, reliable diagnosis from single cells". Methods Mol Med. 132: 65-85. PMID 17876077.
3. ^ a b c d e Saiki et al. "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science vol. 239 pp. 487-91 (1988).
4. ^ Cheng S, Fockler C, Barnes WM, Higuchi R. "Effective amplification of long targets from cloned inserts and human genomic DNA." Proc Natl Acad Sci vol. 91(12) pp. 5695-9 (1994).
5. ^ Q. Chou, M. Russell, D.E. Birch, J. Raymond and W. Bloch (1992). "Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications". Nucleic Acids Research 20: 1717-1723.
6. ^ Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991). "'Touchdown' PCR to circumvent spurious priming during gene amplification.". Nucl Acids Res 19: 4008.
7. ^ Boehnke M et al. "Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm." Am J Hum Genet vol. 45(1) pp. 21-32 (1989).
8. ^ Kwok S et al. "Identification of HIV sequences by using in vitro enzymatic amplification and oligomer cleavage detection." J. Virol. vol. 61(5) pp. 1690-4 (1987).
9. ^ Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides". Gene 164: 49-53. PMID 7590320.
10. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes", in Kieleczawa J: DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett, pp. 241-257. ISBN 0-7637338-3-0.
11. ^ Mueller PR, Wold B (1988). "In vivo footprinting of a muscle specific enhancer by ligation mediated PCR". Science 246: 780-786. PMID 2814500.
12. ^ Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB (1996). "Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands". Proc Natl Acad Sci U S A 93 (13): 9821-9826. PMID 8790415.
13. ^ Longo MC, Berninger MS, Hartley JL "Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions." Gene vol. 93(1) pp. 125-8 (1990).
14. ^ E. Zietkiewicz, A. Rafalski, and D. Labuda (1994). "Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification". Genomics 20 (2): 176-83.
15. ^ Cline J,Braman JC, Hogrefe HH "PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases." Nucleic Acids Research vol. 24(18) pp. 3546-51 (1996).
16. ^ Ochman H, Gerber AS, Hartl DL (1988). "Genetic applications of an inverse polymerase chain reaction". Genetics 120: 621-623. PMID 2852134.
17. ^ Y.G. Liu and R. F. Whittier (1995). "Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking". Genomics 25 (3): 674-81.
18. ^ Myriam Vincent, Yan Xu and Huimin Kong (2004). "Helicase-dependent isothermal DNA amplification". EMBO reports 5 (8): 795–800.
19. ^ David, F.Turlotte, E., (1998). "An Isothermal Amplification Method". C.R.Acad. Sci Paris, Life Science 321 (1): 909-914.
20. ^ Fabrice David (September-October 2002). Utiliser les propriétés topologiques de l’ADN: une nouvelle arme contre les agents pathogènes. Fusion.(in French)
Retrieved from "http://en.wikipedia.org/wiki/Variants_of_PCR"
AFLP PCR
What’s AFLP?-- From Wikipedia, the free encyclopedia
Amplified fragment length polymorphism PCR (or AFLP-PCR or just AFLP) is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic engineering. Developed in the early 1990’s by Keygene, AFLP uses restriction enzymes to cut genomic DNA, followed by ligation of complementary double stranded adaptors to the ends of the restriction fragments. A subset of the restriction fragments are then amplified using two primers complementary to the adaptor and restriction site fragments. The fragments are visualized on denaturing polyacrylamide gels either through autoradiography or fluorescence methodologies.
AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA. The technique was originally described by Vos and Zabeau in 1993. The procedure of this technique is divided into three steps:
1. Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments.
2. Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences.
3. Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band pattern.
A variation on AFLP is cDNA-AFLP, which is used to quantify differences in gene expression levels.
Another variation on AFLP is TE Display, used to detect transposable element mobility.
AFLP
AFLP protocolAFLP protocolAFLP TechnologiesAFLP: principles and applications
AFLPs on the ABI 3100Amplified Fragment Length Polymorphism ProtocolStandard List for AFLP Primer NomenclatureAFLP: not only for fingerprinting, but for positional cloningAFLP PCR
In silico AFLP-PCR amplification
AFLP: not only for Fingerprinting, but for Positional Cloning
GE Healthcare Life Sciences - AFLP
AFLP (Liscum Lab.)
A PCR-based DNA fingerprinting technique: AFLP.
cDNA-AFLP Protocol
AFLP Protocol
References:
AFLP vs PCR-RFLP
AFLP Protocol Using Li-Cor Sequencer
http://www.oznet.ksu.edu/wheatgenotyping/aflp.html
AFLP
Selected links about AFLP. ... Amplified Fragment Length Polymorphisms and Microsatellites: A phylogenetic perspective This paper reviews the techniques of ...
www.pcrlinks.com/variants/aflp.htm
Amplified Fragment Length Polymorphism World of Genetics
Amplified Fragment Length Polymorphism World of Genetics. Amplified Fragment Length Polymorphism summary with 1 pages of encyclopedia entries, ...
www.bookrags.com/.../amplified-fragment-length-polymorph-wog/
Fluorescent Amplified Fragment Length Polymorphism Analysis of ...
Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates. Ticknor LO, Kolst AB, ...
www.ncbi.nlm.nih.gov/pubmed/11571195
Fluorescent amplified fragment length polymorphism analysis of ...
Fluorescent amplified fragment length polymorphism analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis isolates. ...
www.ncbi.nlm.nih.gov/pubmed/14766590
Google Directory - Science > Biology > Biochemistry and Molecular ...
A description of the Amplified Fragment Length Polymorphism technique of DNA fingerprinting. By U Melcher, Oklahoma State University, USA. ...
www.google.com/.../Science/.../Biochemistry_and_Molecular_Biology/.../PCR/....
Genetic Analysis - Amplified Fragment Length Polymorphism
Amplified Fragment Length Polymorphism (AFLP) analysis is one of the most powerful DNA fingerprinting methodologies for the identification and typing of ...
www.beckmancoulter.com/.../applications/.../ceq_aflp_app_stat_dcr.asp...
Amplified fragment length polymorphism based identification of ...
Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies ...
jmm.sgmjournals.org/cgi/content/abstract/54/12/1217
Impact of Amplified Fragment Length Polymorphism Size Homoplasy on ...
Impact of Amplified Fragment Length Polymorphism Size Homoplasy on the Estimation of Population Genetic Diversity and the Detection of Selective Loci ...
www.genetics.org/cgi/content/abstract/179/1/539
A Combined Amplified Fragment Length Polymorphism and Randomly ...
(RAPD) and amplified fragment length polymorphisms. and other tree species (Crous 1998). Septoria tritici. (AFLPs) allows a highly efficient generation of ...
www.genetics.org/cgi/reprint/161/4/1497.pdf
Fluorescence-labeled Methylation-sensitive Amplified Fragment ...
Fluorescence-labeled Methylation-sensitive Amplified Fragment Length Polymorphism (FL-MS-AFLP) Analysis for Quantitative Determination of DNA Methylation ...
jjco.oxfordjournals.org/cgi/content/abstract/38/4/317
Amplified fragment length polymorphism analysis of different ...
eral utility of amplified fragment length polymorphisms. (AFLPs) for molecular analysis of insect genomes. For highly. complex genomes, such as those from ...
journals.cambridge.org/.../action/cjoGetFulltext%3Ffulltextid%3D889168...
Amplified Fragment Length Polymorphisms (AFLPs) detected with non ...
anthracis as detected by Amplified fragment length polymorphism markers. J. Bact. 179:818
pubs.nrc-cnrc.gc.ca/ispmb/ispmb15/15255-3.pdf
K-State Research Exchange: Amplified fragment length polymorphism ...
Title: Amplified fragment length polymorphism in Mycosphaerella graminicola. Authors: Kabbage, Mehdi. Graduation Date: May 2007. Type: Dissertation ...
krex.k-state.edu/dspace/handle/2097/255 [cache]
Amplified-Fragment Length Polymorphism Fingerprinting of ...
Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. ...
jcm.asm.org/cgi/content/abstract/37/10/3300
Using Amplified Fragment Length Polymorphisms and Simple Sequence ...
We have used two DNA-based methods, amplified fragment length polymorphisms (AFLPs) and simple sequence length polymorphisms (SSLPs), to distinguish six ...
crop.scijournals.org/cgi/content/abstract/39/6/1715
Amplified fragment length polymorphisms and sequence data in the ...
4 Phenogram of the neighbor-joining analysis of amplified fragment length polymorphism (AFLP) fingerprints using Nei
www.blackwell-synergy.com/.../pdf/.../j.1469-8137.2007.02172.x
Brazilian Journal of Microbiology - Use of single-enzyme amplified ...
Use of single-enzyme amplified fragment length polymorphism for typing Clostridium ... In the present study amplified fragment length polymorphism analyses ...
www.scielo.br/scielo.php%3Fpid%3DS1517-83822006000300034%26script%3Dsci_art...
BioMed Central Full text Combined use of Amplified Fragment ...
Amplified Fragment Length Polymorphism (AFLP) would scan the entire genome ... Here we evaluate the ability of Amplified Fragment Length Polymorphism (AFLP) ...
www.biomedcentral.com/1471-2334/7/86
Amplified fragment length polymorphism of Streptococcus suis ...
Amplified fragment length polymorphism of Streptococcus suis strains correlates with their profile of virulence-associated genes and clinical background ...
jmm.sgmjournals.org/cgi/content/abstract/56/1/102
Chromosome numbers and pollen stainability of three species of ...
This Article. Right arrow, Full Text. Right arrow, Full Text (PDF). Right arrow, Submit a response. Right arrow, Alert me when this article is cited ...
www.amjbot.org/cgi/content/abstract/88/4/693
Amplified Fragment Length Polymorphism Analysis of Campylobacter ...
The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships between ...
aem.asm.org/cgi/content/abstract/66/9/3917
Amplified Fragment Length Polymorphism (AFLP) Provides Molecular ...
Amplified fragment length polymorphism (AFLP) analysis using 17 primer combinations was carried out on two species of Caladium (C. bicolor and C. ...
aob.oxfordjournals.org/cgi/content/abstract/84/2/155
Springer Protocols: Abstract: Amplified Fragment Length ...
Amplified Fragment Length Polymorphism Analysis of Salmonella enterica ... Amplified fragment length polymorphism (AFLP) is a powerful PCR-based ...
www.springerprotocols.com/.../doi/.../978-1-59745-512-1_8
Amplified fragment length polymorphism PCR (or AFLP-PCR or just AFLP) is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic engineering. Developed in the early 1990’s by Keygene, AFLP uses restriction enzymes to cut genomic DNA, followed by ligation of complementary double stranded adaptors to the ends of the restriction fragments. A subset of the restriction fragments are then amplified using two primers complementary to the adaptor and restriction site fragments. The fragments are visualized on denaturing polyacrylamide gels either through autoradiography or fluorescence methodologies.
AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA. The technique was originally described by Vos and Zabeau in 1993. The procedure of this technique is divided into three steps:
1. Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments.
2. Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences.
3. Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band pattern.
A variation on AFLP is cDNA-AFLP, which is used to quantify differences in gene expression levels.
Another variation on AFLP is TE Display, used to detect transposable element mobility.
AFLP
AFLP protocolAFLP protocolAFLP TechnologiesAFLP: principles and applications
AFLPs on the ABI 3100Amplified Fragment Length Polymorphism ProtocolStandard List for AFLP Primer NomenclatureAFLP: not only for fingerprinting, but for positional cloningAFLP PCR
In silico AFLP-PCR amplification
AFLP: not only for Fingerprinting, but for Positional Cloning
GE Healthcare Life Sciences - AFLP
AFLP (Liscum Lab.)
A PCR-based DNA fingerprinting technique: AFLP.
cDNA-AFLP Protocol
AFLP Protocol
References:
AFLP vs PCR-RFLP
AFLP Protocol Using Li-Cor Sequencer
http://www.oznet.ksu.edu/wheatgenotyping/aflp.html
AFLP
Selected links about AFLP. ... Amplified Fragment Length Polymorphisms and Microsatellites: A phylogenetic perspective This paper reviews the techniques of ...
www.pcrlinks.com/variants/aflp.htm
Amplified Fragment Length Polymorphism World of Genetics
Amplified Fragment Length Polymorphism World of Genetics. Amplified Fragment Length Polymorphism summary with 1 pages of encyclopedia entries, ...
www.bookrags.com/.../amplified-fragment-length-polymorph-wog/
Fluorescent Amplified Fragment Length Polymorphism Analysis of ...
Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates. Ticknor LO, Kolst AB, ...
www.ncbi.nlm.nih.gov/pubmed/11571195
Fluorescent amplified fragment length polymorphism analysis of ...
Fluorescent amplified fragment length polymorphism analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis isolates. ...
www.ncbi.nlm.nih.gov/pubmed/14766590
Google Directory - Science > Biology > Biochemistry and Molecular ...
A description of the Amplified Fragment Length Polymorphism technique of DNA fingerprinting. By U Melcher, Oklahoma State University, USA. ...
www.google.com/.../Science/.../Biochemistry_and_Molecular_Biology/.../PCR/....
Genetic Analysis - Amplified Fragment Length Polymorphism
Amplified Fragment Length Polymorphism (AFLP) analysis is one of the most powerful DNA fingerprinting methodologies for the identification and typing of ...
www.beckmancoulter.com/.../applications/.../ceq_aflp_app_stat_dcr.asp...
Amplified fragment length polymorphism based identification of ...
Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies ...
jmm.sgmjournals.org/cgi/content/abstract/54/12/1217
Impact of Amplified Fragment Length Polymorphism Size Homoplasy on ...
Impact of Amplified Fragment Length Polymorphism Size Homoplasy on the Estimation of Population Genetic Diversity and the Detection of Selective Loci ...
www.genetics.org/cgi/content/abstract/179/1/539
A Combined Amplified Fragment Length Polymorphism and Randomly ...
(RAPD) and amplified fragment length polymorphisms. and other tree species (Crous 1998). Septoria tritici. (AFLPs) allows a highly efficient generation of ...
www.genetics.org/cgi/reprint/161/4/1497.pdf
Fluorescence-labeled Methylation-sensitive Amplified Fragment ...
Fluorescence-labeled Methylation-sensitive Amplified Fragment Length Polymorphism (FL-MS-AFLP) Analysis for Quantitative Determination of DNA Methylation ...
jjco.oxfordjournals.org/cgi/content/abstract/38/4/317
Amplified fragment length polymorphism analysis of different ...
eral utility of amplified fragment length polymorphisms. (AFLPs) for molecular analysis of insect genomes. For highly. complex genomes, such as those from ...
journals.cambridge.org/.../action/cjoGetFulltext%3Ffulltextid%3D889168...
Amplified Fragment Length Polymorphisms (AFLPs) detected with non ...
anthracis as detected by Amplified fragment length polymorphism markers. J. Bact. 179:818
pubs.nrc-cnrc.gc.ca/ispmb/ispmb15/15255-3.pdf
K-State Research Exchange: Amplified fragment length polymorphism ...
Title: Amplified fragment length polymorphism in Mycosphaerella graminicola. Authors: Kabbage, Mehdi. Graduation Date: May 2007. Type: Dissertation ...
krex.k-state.edu/dspace/handle/2097/255 [cache]
Amplified-Fragment Length Polymorphism Fingerprinting of ...
Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. ...
jcm.asm.org/cgi/content/abstract/37/10/3300
Using Amplified Fragment Length Polymorphisms and Simple Sequence ...
We have used two DNA-based methods, amplified fragment length polymorphisms (AFLPs) and simple sequence length polymorphisms (SSLPs), to distinguish six ...
crop.scijournals.org/cgi/content/abstract/39/6/1715
Amplified fragment length polymorphisms and sequence data in the ...
4 Phenogram of the neighbor-joining analysis of amplified fragment length polymorphism (AFLP) fingerprints using Nei
www.blackwell-synergy.com/.../pdf/.../j.1469-8137.2007.02172.x
Brazilian Journal of Microbiology - Use of single-enzyme amplified ...
Use of single-enzyme amplified fragment length polymorphism for typing Clostridium ... In the present study amplified fragment length polymorphism analyses ...
www.scielo.br/scielo.php%3Fpid%3DS1517-83822006000300034%26script%3Dsci_art...
BioMed Central Full text Combined use of Amplified Fragment ...
Amplified Fragment Length Polymorphism (AFLP) would scan the entire genome ... Here we evaluate the ability of Amplified Fragment Length Polymorphism (AFLP) ...
www.biomedcentral.com/1471-2334/7/86
Amplified fragment length polymorphism of Streptococcus suis ...
Amplified fragment length polymorphism of Streptococcus suis strains correlates with their profile of virulence-associated genes and clinical background ...
jmm.sgmjournals.org/cgi/content/abstract/56/1/102
Chromosome numbers and pollen stainability of three species of ...
This Article. Right arrow, Full Text. Right arrow, Full Text (PDF). Right arrow, Submit a response. Right arrow, Alert me when this article is cited ...
www.amjbot.org/cgi/content/abstract/88/4/693
Amplified Fragment Length Polymorphism Analysis of Campylobacter ...
The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships between ...
aem.asm.org/cgi/content/abstract/66/9/3917
Amplified Fragment Length Polymorphism (AFLP) Provides Molecular ...
Amplified fragment length polymorphism (AFLP) analysis using 17 primer combinations was carried out on two species of Caladium (C. bicolor and C. ...
aob.oxfordjournals.org/cgi/content/abstract/84/2/155
Springer Protocols: Abstract: Amplified Fragment Length ...
Amplified Fragment Length Polymorphism Analysis of Salmonella enterica ... Amplified fragment length polymorphism (AFLP) is a powerful PCR-based ...
www.springerprotocols.com/.../doi/.../978-1-59745-512-1_8
Alu-PCR
Alu-PCR amplificationUniversity of Bari, Italy
Alu-PCR hybridizationFondation Jean Dausset
Gene Connection
Protocol for Detection of Alu by PCR
3' Alu PCR: a simple and rapid method to isolate human polymorphic markers
http://nar.oxfordjournals.org/cgi/content/abstract/20/6/1333
FISH guide - Alu-PCR, Alu-banding (Harvard)
Alu-PCR hybridizationFondation Jean Dausset
Gene Connection
Protocol for Detection of Alu by PCR
3' Alu PCR: a simple and rapid method to isolate human polymorphic markers
http://nar.oxfordjournals.org/cgi/content/abstract/20/6/1333
FISH guide - Alu-PCR, Alu-banding (Harvard)
Asymmetric PCR
What is asymmetric PCR? A PCR in which the predominant product is a single-stranded DNA, as a result of unequal primer concentrations. As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into double-stranded DNA. The higher concentration primer continues to primer synthesis, but only of its strand.
Asymmetric PCR Protocol 1. Pick a phage plaque and place in 100 ul TE or scrape a fresh colony of a bacterial transformant of choice and place in 50 ul of TE/TX100 in a microcentrifuge tube. 2. Heat the tube for 10 min at 95C. 3. Centrifuge at maximum speed for several minutes in a microcentrifuge to pellet cell debris. Collect the supernatant. 4. Add the following components in a PCR tube: 5 ul of phage or bacterial extract (from Step #A3) 50 uM of dNTPs 50 pmol of Primer 1 1 pmol of Primer 2 in 1X PCR Reaction Buffer to give a final reaction volume of 50 to 100 ul 2.5 Units of Taq polymerase 5. Run 30 to 35 cycles in a thermocycler using the following PCR program (see Hint #1 and #2) 95C for 60 sec 60C for 30 sec 72C for 2 min 6. Run a small aliquot on an agarose gel to analyze for single-stranded DNA (see Protocol on Agarose Gel Electrophoresis of DNA). 7. Purify the PCR products and sequence, if desired.
Asymmetric PCR for ssDNA ProductionHOT ASYMMETRIC PCR (Mullins Lab)
Direct sequencing by thermal asymmetric PCR
Rapid sequencing of unpurified PCR products by thermal asymmetric PCR cycle sequencing using unlabeled sequencing primers.
Detection of asymmetric PCR products in homogeneoussolution by fluorescence correlation spectroscopy
Asymmetric PCR Using the Primers Anchored on the surface of magnetic nanoparticles.
Asymmetric PCR Protocol 1. Pick a phage plaque and place in 100 ul TE or scrape a fresh colony of a bacterial transformant of choice and place in 50 ul of TE/TX100 in a microcentrifuge tube. 2. Heat the tube for 10 min at 95C. 3. Centrifuge at maximum speed for several minutes in a microcentrifuge to pellet cell debris. Collect the supernatant. 4. Add the following components in a PCR tube: 5 ul of phage or bacterial extract (from Step #A3) 50 uM of dNTPs 50 pmol of Primer 1 1 pmol of Primer 2 in 1X PCR Reaction Buffer to give a final reaction volume of 50 to 100 ul 2.5 Units of Taq polymerase 5. Run 30 to 35 cycles in a thermocycler using the following PCR program (see Hint #1 and #2) 95C for 60 sec 60C for 30 sec 72C for 2 min 6. Run a small aliquot on an agarose gel to analyze for single-stranded DNA (see Protocol on Agarose Gel Electrophoresis of DNA). 7. Purify the PCR products and sequence, if desired.
Asymmetric PCR for ssDNA ProductionHOT ASYMMETRIC PCR (Mullins Lab)
Direct sequencing by thermal asymmetric PCR
Rapid sequencing of unpurified PCR products by thermal asymmetric PCR cycle sequencing using unlabeled sequencing primers.
Detection of asymmetric PCR products in homogeneoussolution by fluorescence correlation spectroscopy
Asymmetric PCR Using the Primers Anchored on the surface of magnetic nanoparticles.
Colony PCR Protocols
Colony PCR protocol
Detailed protocol from the web site of the Department of Biology, University of Michigan, USA.www.mcdb.lsa.umich.edu/labs/maddock/protocols/PCR/colony_pcr.html
Colony PCRColony PCR. This protocol is designed to quickly screen for plasmid inserts directly from E. coli. colonies. The plasmid should be high copy number such as ...www.csun.edu/~mls42367/Protocols/Colony%20PCR.pdf
Colony PCR ProtocolColony PCR Protocol. 1. Pull out eight glycerol stock plates from the –80. o. C freezer and set on bench top to. thaw. Be sure to remove the foil seal ...microarrays.berkeley.edu/file_download.php?fid=13
Colony PCR ProtocolColony PCR Protocol. Overnight Culture 5.0µl. 10x PCR buffer 5.0µl. MgCl2 (25mM) 5.0µl. Forward primer (10µM) 0.2µl. Reverse primer (10µM) 0.2µl ...www.unc.edu/~fconlon/Protocols/Colony%20PCR%20Protocol.doc
Colony PCR
Colony PCR Protocol contributed by Lynn Hancock 1. Choose a reasonable sized colony (2-3 mm in diameter) and resuspend 100 µl ddH2O. 2. For PCR, ...www.enterococcus.ouhsc.edu/ColonyPCRProtocol.asp
Yeast Colony PCR -- Amberg et al.
Search CSH Protocols. Advanced Search · Find Protocols · Find a Kit · Access Personal Page · Submit Protocols · Help · Subscribe · About CSH Protocols ...www.cshprotocols.org/cgi/content/short/2006/1/pdb.prot4170
Colony PCR - OpenWetWare
From OpenWetWare. Jump to: navigation, search. See Colony PCR for general information about this protocol and other variants ...openwetware.org/wiki/Endy:Colony_PCR
Colony PCR protocol
Monserate Biotechnology Group Custom services, Colony PCR protocol.www.monseratebiotech.com/colony-pcr.html
Yeast Colony PCR protocolYeast Colony PCR protocol. derived from http://sequence-. www.stanford.edu/group/yeast_deletion_project/deletions3.html.
Colony PCR (Forsburg Lab)
The Forsburg lab pages: fission yeast colony PCR protocol. ... Colony PCR can be used to identify colonies where your favorite gene yfg1 has been replaced ...http://www-rcf.usc.edu/~forsburg/pcr.html
Rapid Screening by Direct Colony PCR Using the FastStart PCR Master
The FastStart PCR Master is a convenient and ideal tool for direct colony PCR. Intact bacteria can be analyzed directly without prior template purification, ...www.analytica-world.com/articles/e/65346/
Detailed protocol from the web site of the Department of Biology, University of Michigan, USA.www.mcdb.lsa.umich.edu/labs/maddock/protocols/PCR/colony_pcr.html
Colony PCRColony PCR. This protocol is designed to quickly screen for plasmid inserts directly from E. coli. colonies. The plasmid should be high copy number such as ...www.csun.edu/~mls42367/Protocols/Colony%20PCR.pdf
Colony PCR ProtocolColony PCR Protocol. 1. Pull out eight glycerol stock plates from the –80. o. C freezer and set on bench top to. thaw. Be sure to remove the foil seal ...microarrays.berkeley.edu/file_download.php?fid=13
Colony PCR ProtocolColony PCR Protocol. Overnight Culture 5.0µl. 10x PCR buffer 5.0µl. MgCl2 (25mM) 5.0µl. Forward primer (10µM) 0.2µl. Reverse primer (10µM) 0.2µl ...www.unc.edu/~fconlon/Protocols/Colony%20PCR%20Protocol.doc
Colony PCR
Colony PCR Protocol contributed by Lynn Hancock 1. Choose a reasonable sized colony (2-3 mm in diameter) and resuspend 100 µl ddH2O. 2. For PCR, ...www.enterococcus.ouhsc.edu/ColonyPCRProtocol.asp
Yeast Colony PCR -- Amberg et al.
Search CSH Protocols. Advanced Search · Find Protocols · Find a Kit · Access Personal Page · Submit Protocols · Help · Subscribe · About CSH Protocols ...www.cshprotocols.org/cgi/content/short/2006/1/pdb.prot4170
Colony PCR - OpenWetWare
From OpenWetWare. Jump to: navigation, search. See Colony PCR for general information about this protocol and other variants ...openwetware.org/wiki/Endy:Colony_PCR
Colony PCR protocol
Monserate Biotechnology Group Custom services, Colony PCR protocol.www.monseratebiotech.com/colony-pcr.html
Yeast Colony PCR protocolYeast Colony PCR protocol. derived from http://sequence-. www.stanford.edu/group/yeast_deletion_project/deletions3.html.
Colony PCR (Forsburg Lab)
The Forsburg lab pages: fission yeast colony PCR protocol. ... Colony PCR can be used to identify colonies where your favorite gene yfg1 has been replaced ...http://www-rcf.usc.edu/~forsburg/pcr.html
Rapid Screening by Direct Colony PCR Using the FastStart PCR Master
The FastStart PCR Master is a convenient and ideal tool for direct colony PCR. Intact bacteria can be analyzed directly without prior template purification, ...www.analytica-world.com/articles/e/65346/
Competitive and/or Quantitative RT-PCR
Competitive RT-PCR (Dieter Kaufmann Lab.)
Quantitative RT-PCR (Morimoto Lab.)
Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures (Jack Vanden Heuvel Lab.)
http://www.cas.psu.edu/docs/CASDEPT/VET/jackvh/jvhpcr.html
Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample (Biological Procedures Online)
http://www.biologicalprocedures.com/bpo/arts/1/20/m20.htm
Quantitative Measurement of mRNA by Competitive RT-PCR. (Springer Protocols)
Improved quantitative real-time RT–PCR for expression profiling of Individual Cells. (Nucleic Acid Research)
Competitive Quantitative RT-PCR (Ambion)
Comparative Study of Different Standardization Concepts in Quantitative Competitive Reverse Transcription-PCR Assays. (JCM)
Procedure for Competitive Quantitative Reverse Transcription PCR. (Transgenomic)
Quantitative RT-PCR (Morimoto Lab.)
Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures (Jack Vanden Heuvel Lab.)
http://www.cas.psu.edu/docs/CASDEPT/VET/jackvh/jvhpcr.html
Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample (Biological Procedures Online)
http://www.biologicalprocedures.com/bpo/arts/1/20/m20.htm
Quantitative Measurement of mRNA by Competitive RT-PCR. (Springer Protocols)
Improved quantitative real-time RT–PCR for expression profiling of Individual Cells. (Nucleic Acid Research)
Competitive Quantitative RT-PCR (Ambion)
Comparative Study of Different Standardization Concepts in Quantitative Competitive Reverse Transcription-PCR Assays. (JCM)
Procedure for Competitive Quantitative Reverse Transcription PCR. (Transgenomic)
Degenerate PCR
The identification of novel members of gene families by PCR using degenerate primers is described and protocols given. Article by Michael Koelle 1996 on the ...www.dartmouth.edu/~ambros/protocols/other/koelle/degenerate_PCR.html
Degenerate PCR.html (Michael Koelle)
by Michael Koelle. The identification of novel members of gene families by PCR using degenerate primers has been considered more of an art than a science, ...www.med.yale.edu/mbb/koelle/protocols/protocol_degenerate_PCR.html
PCR Primer Design
However, I have used primers with as high as 256- and 1024-fold degeneracy for the successful amplification and subsequent direct sequencing of a wide range ...www.mcb.uct.ac.za/pcroptim.htm
Degenerate PCR (NTNU Cell and Molecular Biology Group)
X - Services · Open Positions, Degenerate PCR Degenerate PCR is in most respects identical to ordinary PCR, but with one major difference. ...http://boneslab.bio.ntnu.no/degpcrshortguide.htm
Designing degenerate PCR primers
The purpose of CODEHOP is to help in the design of degenerate PCR primers from protein sequence. Given a multiple alignment of a series of target proteins, ...genomebiology.com/2000/1/1/reports/240
Nature Protocols: Quantitative multiplex degenerate PCR for human endogenous retrovirus expression profiling.
Last, the protocol below provides general rules for the design of MD-PCR applications. Once primers have been designed and optimized, the procedure can be ...www.natureprotocols.com/2007/01/25/quantitative_multiplex_degener.php
In Situ PCR
The In Situ PCR:
(Amplification and Detection in a Cellular Context)
Ernest F. Retzel, Katherine A. Staskus, Janet E. Embretson and Ashley T. Haase
Department of Microbiology, University of Minnesota, Minneapolis, MN 55455
Table of Contents
Introduction Technical Aspects/Philosophy Primer Design Reaction Conditions "Mechanics" of the Reaction Methodology
A. Cell and Tissues.
1. Cultured Cells: Infection and Preparation. 2. Tissues: Pulmonary infection and preparation.
B. In Situ Amplification
1. Cultured Cells in Suspension. 2. Tissues on Slides.
C. Detection of Amplified DNA by in situ Hybridization
1. Solution amplification. 2. Tissue Sections.
References
In Situ PCR on Plant Material (Bo Johansen)Required materialsRequired solutionsTissue preparationPectinase treatmentProtease treatmentAcetylationDNase treatmentReverse transcriptionThermal cyclingDetectionMountingAcrobat version of PosterReferencesOther ISPCR linksBotanical Institute
RT IN SITU PCR
Gerard J. Nuovo
TABLE OF CONTENTS
1. Abstract
2. Introductory statement
3. The key preparatory steps
3.1. Fixative
3.1.1. Protease digestion
3.1.2. Definition of optimal protease digestion
3.1.3. Definitions of suboptimal and over-digestion
3.1.4. DNase digestion
3.1.5. Direct incorporation of the reporter nucleotide
4. A one step protocol for RT in situ PCR
5. Applications of RT in situ PCR Matrix metalloprotease expression in cancer as a model for RT in situ PCR
5.1. MMP and TIMP expression in cervical cancer
6. Concluding remarks
7. References
8. Entire manuscript
In situ PCR: protocols and applications.
groups have published protocols and data using in situ PCR techniques. (5). This article will discuss some of the basic concepts of in situ PCR, the proto- ...www.genome.org/cgi/reprint/4/4/S151.pdf
Co-labeling Using In Situ PCR: A Review
Figure 2 Background vs signal with RT in situ PCR. This renal biopsy was analyzed for mRNA by RT in situ PCR. After digestion for 10 min in ...www.jhc.org/cgi/reprint/49/11/1329.pdf
Protocols for the : in situ: PCR-amplification and detection of mRNA and DNA sequencing.
In this protocol we describe the in situ PCR method for the amplification of both DNA and mRNA targets [in situ reverse transcriptase-PCR (RT-PCR)], ...www.nature.com/nprot/journal/v2/n11/abs/nprot.2007.395.html
96-well RNA In Situ Hybridization Protocol
Materials. 1.1. Probe Preparation. 1.1.1. Cell Inoculation. 1. 96-well, deep square-well round bottom plate (E&K Scientific Ritter Riplate). ...www.fruitfly.org/about/methods/RNAinsitu.html
A protocol for PCR in situ hybridization of hyphomycetesIntroduction. Within the last years the focus of mycological studies has shifted. emphasis from mere taxonomic studies to more ecological. questions. ...www.im.microbios.org/03setember98/08%20Sterflinger.pdf
(Amplification and Detection in a Cellular Context)
Ernest F. Retzel, Katherine A. Staskus, Janet E. Embretson and Ashley T. Haase
Department of Microbiology, University of Minnesota, Minneapolis, MN 55455
Table of Contents
Introduction Technical Aspects/Philosophy Primer Design Reaction Conditions "Mechanics" of the Reaction Methodology
A. Cell and Tissues.
1. Cultured Cells: Infection and Preparation. 2. Tissues: Pulmonary infection and preparation.
B. In Situ Amplification
1. Cultured Cells in Suspension. 2. Tissues on Slides.
C. Detection of Amplified DNA by in situ Hybridization
1. Solution amplification. 2. Tissue Sections.
References
In Situ PCR on Plant Material (Bo Johansen)Required materialsRequired solutionsTissue preparationPectinase treatmentProtease treatmentAcetylationDNase treatmentReverse transcriptionThermal cyclingDetectionMountingAcrobat version of PosterReferencesOther ISPCR linksBotanical Institute
RT IN SITU PCR
Gerard J. Nuovo
TABLE OF CONTENTS
1. Abstract
2. Introductory statement
3. The key preparatory steps
3.1. Fixative
3.1.1. Protease digestion
3.1.2. Definition of optimal protease digestion
3.1.3. Definitions of suboptimal and over-digestion
3.1.4. DNase digestion
3.1.5. Direct incorporation of the reporter nucleotide
4. A one step protocol for RT in situ PCR
5. Applications of RT in situ PCR Matrix metalloprotease expression in cancer as a model for RT in situ PCR
5.1. MMP and TIMP expression in cervical cancer
6. Concluding remarks
7. References
8. Entire manuscript
In situ PCR: protocols and applications.
groups have published protocols and data using in situ PCR techniques. (5). This article will discuss some of the basic concepts of in situ PCR, the proto- ...www.genome.org/cgi/reprint/4/4/S151.pdf
Co-labeling Using In Situ PCR: A Review
Figure 2 Background vs signal with RT in situ PCR. This renal biopsy was analyzed for mRNA by RT in situ PCR. After digestion for 10 min in ...www.jhc.org/cgi/reprint/49/11/1329.pdf
Protocols for the : in situ: PCR-amplification and detection of mRNA and DNA sequencing.
In this protocol we describe the in situ PCR method for the amplification of both DNA and mRNA targets [in situ reverse transcriptase-PCR (RT-PCR)], ...www.nature.com/nprot/journal/v2/n11/abs/nprot.2007.395.html
96-well RNA In Situ Hybridization Protocol
Materials. 1.1. Probe Preparation. 1.1.1. Cell Inoculation. 1. 96-well, deep square-well round bottom plate (E&K Scientific Ritter Riplate). ...www.fruitfly.org/about/methods/RNAinsitu.html
A protocol for PCR in situ hybridization of hyphomycetesIntroduction. Within the last years the focus of mycological studies has shifted. emphasis from mere taxonomic studies to more ecological. questions. ...www.im.microbios.org/03setember98/08%20Sterflinger.pdf
Differential Display PCR
About Differential Display PCR: All living organisms have thousands to tens of thousands of unique genes encoded in their genome, of which only a small fraction, perhaps 15%, are expressed in any individual cell. Therefore, it is the temporal and spatial regulation in gene expression that determines life processes. The course of normal cellular development as well as pathological changes that arise in diseases such as cancer are all believed to be driven by changes in gene expression. A pressing problem is to identify and characterize those genes that are differentially expressed in order to understand the molecular nature of disease state and subsequently, to devise rational therapies. Differential Display was invented in 1992 by Drs. Arthur Pardee and Peng Liang to allow rapid, accurate and sensitive detection of altered gene expression (Science. 1992, 257:967; U.S. Patent 5,262,311).
Further readings about differential display technique
What's Differential Display (GenHunter)Introduction to differential display technique
Differential Display (Chun-Ming Liu)The following procedures are described:
RNA extraction and qualification
DNase treatment of RNA sample
Reverse transcription
PCR amplification
Separation on acrylamide gels
DNA extraction from bands of interest
Re-amplification by PCR
Separation on agarose gels
Excision of amplified products
Differential Display (Breeden Lab)Detailed protocol for differential display
Differential Display (Plant Molecular Biolgy Lab)It's for plant RNA display. The protocol should be general.
Differential Display-Reverse Transcription-PCR (Gerard Lazo)
Rational primer design greatly improves differential display-PCR.
Applications of Differential-Display Reverse Transcription-PCR.
Differential Display-PCR -- Sambrook and Russell.
Development and optimization of a fluorescent differential display.
The Science Advisory Board – Differential Display PCR.
Perspective: Micoarrays and Differential Display PCR.
Laboratory Techniques: Differential Display - Polymerase Chain Reaction
Effect of Primer Purity on the Banding Patterns of DifferentialDisplay Polymerase Chain ReactionDifferential Display of RNAs (Breeden Lab.)
Further readings about differential display technique
What's Differential Display (GenHunter)Introduction to differential display technique
Differential Display (Chun-Ming Liu)The following procedures are described:
RNA extraction and qualification
DNase treatment of RNA sample
Reverse transcription
PCR amplification
Separation on acrylamide gels
DNA extraction from bands of interest
Re-amplification by PCR
Separation on agarose gels
Excision of amplified products
Differential Display (Breeden Lab)Detailed protocol for differential display
Differential Display (Plant Molecular Biolgy Lab)It's for plant RNA display. The protocol should be general.
Differential Display-Reverse Transcription-PCR (Gerard Lazo)
Rational primer design greatly improves differential display-PCR.
Applications of Differential-Display Reverse Transcription-PCR.
Differential Display-PCR -- Sambrook and Russell.
Development and optimization of a fluorescent differential display.
The Science Advisory Board – Differential Display PCR.
Perspective: Micoarrays and Differential Display PCR.
Laboratory Techniques: Differential Display - Polymerase Chain Reaction
Effect of Primer Purity on the Banding Patterns of DifferentialDisplay Polymerase Chain ReactionDifferential Display of RNAs (Breeden Lab.)
Inverse PCR
Inverse PCR & Cycle Sequencing of P Element Insertions for STS GenerationJay Rehm
Inverse PCR for PAC-end sequencingBrad Barbazuk
Inverse PCR:For use with Snyder mTn-lacZ/LEU2 based mutagenesisFred Hutchinson Cancer Research Center
Cloning flanking DNA from pD991 enhancer trap T-DNA insertsDepartment of Biological Sciences Dartmouth College
Inverse PCR protocol
Maddock Lab.
Inverse PCR and Sequencing Protocol
E. Jay Rehm
Inverse PCR
Trevor Epp
Springer Protocols: Inverse PCR (IPCR) for Obtaining Promoter Sequence.
Springer Protocols
Inverse PCR for PAC-end sequencingBrad Barbazuk
Inverse PCR:For use with Snyder mTn-lacZ/LEU2 based mutagenesisFred Hutchinson Cancer Research Center
Cloning flanking DNA from pD991 enhancer trap T-DNA insertsDepartment of Biological Sciences Dartmouth College
Inverse PCR protocol
Maddock Lab.
Inverse PCR and Sequencing Protocol
E. Jay Rehm
Inverse PCR
Trevor Epp
Springer Protocols: Inverse PCR (IPCR) for Obtaining Promoter Sequence.
Springer Protocols
Ligation Mediated Suppression PCR
(adapted from McKinney et al., 1995, Siebert, 1995, Strauss et al., 2001 and Alonso et al., 2003)
PURPOSE: To analyze unknown flanking genomic sequences adjacent to a T-DNA left border
1.) Isolation of DNA
-collect 2-3 young leaves in an eppendorf tube
-add 100 uL extraction buffer and add proteinase K, grind tissue using a blue pestel (no large pieces of leaf should be left).
-add another 100 uL of extraction buffer, vortex, and incubate in 37C for 30 min.
-add 200 uL of saturated phenol and vortex.
-spin at max speed in centrifuge for 2 min.
-collect upper phase to new eppendorf tube.
-add 200 uL of (24:1) chloroform:isoamyl alcohol, vortex, centrifuge at max speed for 2 min.
-collect upper phase into a new eppendorf tube.
-add 18 uL of 3M sodium acetate and add 400 uL of 100% EtOH, mix by inverting and incubate for 10 min at 4C.
-spin in centrifuge at max speed for 10 min.
-pour supernatant off and wash with 500 uL of 70% EtOH
-spin in centrifuge at max speed for 5 min.
-pour supernatant off and wash again with 500 uL of 70% EtOH
-spin in centrifuge at max speed for 5 min.
-pour off supernatant
-carefully pipette off excess EtOH
-let pellet dry for 45 min in the hood.
-resuspend DNA in 100 uL of TE
-store in -20C
2.) Digestion
-mix together:
50 uL gDNA (from above)
10 uL 10x Buffer 2
1 uL Hind III
39 uL dH2O
TOTAL volume 100 uL
-digest overnight at 37C
3.) Digestion Clean Up
-heat inactivate at 65C for 20 min.
-add 100 µL of chloroform
-mix by inverting tubes
-spin in centrifuge at max speed for 5 min.
-collect upper phase into a new eppendorf tube with 200 uL of isopropanol
-mix by inverting tubes
-incubate at room temperature for 10 min
-spin at max speed for 10 min
-remove supernatant
-wash with 100 uL of 70% EtOH
-spin at max speed for 5 min
-remove supernatant
-dry in hood for 45 min.
-resuspend in 20 uL of dH2O
4.) Constructing adapters for ligation
*adapters for ligation to Hind III ends are made by annealing oligos ADAPS-E1(5’-aattcacctgcccgg/3AmMc7/-3’) w/ a 3’ amino terminal end and ADAPL-E1(5’-ctaatacgactcactatagggctcgagcggccgcccgggcaggtg-3’). Oligos may be purchased from IDT at www.idtdna.com
-dilute ADAPS and ADAPL to 100uM
-combine in equal amts of ADAPS and ADAPL (i.e. add 10 µL of ADAPS add 10 uL of ADAPL)
-vortex briefly
-place tube in 500 mL of boiling H2O for 2 min
-remove heat and let bath cool for 1 hr. (this is to ensure correct nucleotide pairing)
-store adapter at -20C
5.) Construction of Adapter Library (ligate adapter to digestion)
-mix together:
10 uL cleaned gDNA digestion
1 uL Adapter (100uM)
2 uL T4 ligase (NEB product)
2 uL 10 x T4 ligase buffer (NEB buffer)
5 uL dH2O
TOTAL volume 20 uL
-vortex and incubate at 16C overnight in thermocycler
-heat inactivate at 65 for 20 min
-add 180 uL of TE (this is your adapter library store at -20C)
6.) Primers for 1º and 2º PCR
*Primary products are generated from amplifying primers AP1 (5’-ggatcctaatacgactcactataggc-3’) and PgwLat52LB-WP1 (5’-ctatgttactagatcgaccgg-3’).
*Secondary products are generated by diluting primary products by 50 fold and amplifying with primers AP2 (5’-tatagggctcgagcggccg-3’) and PgwLat52LB-WP2 (5’-caattcggcgttaattcagtac-3’).
-Primers come from IDT and must be diluted to 10 uM concentration before using.
7.) Primary PCR
-mix together:
0.125 uL Ex Taq (Takara)
2.5 uL 10 x Ex Taq Buffer
2.0 uL dNTP Mix
1.0 uL AP1
1.0 uL WP1
17.375 uL dH20
1.0 uL Adapter Library
TOTAL volume 25 µL
*Run on LMS_PCR2 (conditions recommended by Takara)
1.) incubate at 94C for 2 min
2.) incubate at 94C for 30 sec
3.) incubate at 55C for 30 sec
4.) incubate at 72C for 1 min
5.) recycle to step 2 for 29 more times
6.) incubate at 65C for 10 min
7.) hold at 4C forever
8.) Secondary PCR
*Dilute primary PCR:
-98 uL of dH20
-2 uL of primary PCR
*Rxn is setup exactly like primary PCR.
9.) Run on 1% Agarose Gel
5g Agarose Low
500mL 1 x TAE
50uL EtBr
*Always run a ladder to verify our bands are between 200 and 2000bp. Should only sequence the lines that give a clear band.
10.)Sequencing
*Need to clean up PCR rxn using Qiagen or Eppendorf kits. Then setup the rxn to be sequenced.
- mix together:
2 uL cleaned secondary PCR
1 uL secondary primer (WP2)
17 uL dH20
TOTAL volume 20 uL
*Sequencing is done through UNC Lineberger Comprehensive Cancer Center (located on campus), and normal turn around time can range from 1-7days.
PURPOSE: To analyze unknown flanking genomic sequences adjacent to a T-DNA left border
1.) Isolation of DNA
-collect 2-3 young leaves in an eppendorf tube
-add 100 uL extraction buffer and add proteinase K, grind tissue using a blue pestel (no large pieces of leaf should be left).
-add another 100 uL of extraction buffer, vortex, and incubate in 37C for 30 min.
-add 200 uL of saturated phenol and vortex.
-spin at max speed in centrifuge for 2 min.
-collect upper phase to new eppendorf tube.
-add 200 uL of (24:1) chloroform:isoamyl alcohol, vortex, centrifuge at max speed for 2 min.
-collect upper phase into a new eppendorf tube.
-add 18 uL of 3M sodium acetate and add 400 uL of 100% EtOH, mix by inverting and incubate for 10 min at 4C.
-spin in centrifuge at max speed for 10 min.
-pour supernatant off and wash with 500 uL of 70% EtOH
-spin in centrifuge at max speed for 5 min.
-pour supernatant off and wash again with 500 uL of 70% EtOH
-spin in centrifuge at max speed for 5 min.
-pour off supernatant
-carefully pipette off excess EtOH
-let pellet dry for 45 min in the hood.
-resuspend DNA in 100 uL of TE
-store in -20C
2.) Digestion
-mix together:
50 uL gDNA (from above)
10 uL 10x Buffer 2
1 uL Hind III
39 uL dH2O
TOTAL volume 100 uL
-digest overnight at 37C
3.) Digestion Clean Up
-heat inactivate at 65C for 20 min.
-add 100 µL of chloroform
-mix by inverting tubes
-spin in centrifuge at max speed for 5 min.
-collect upper phase into a new eppendorf tube with 200 uL of isopropanol
-mix by inverting tubes
-incubate at room temperature for 10 min
-spin at max speed for 10 min
-remove supernatant
-wash with 100 uL of 70% EtOH
-spin at max speed for 5 min
-remove supernatant
-dry in hood for 45 min.
-resuspend in 20 uL of dH2O
4.) Constructing adapters for ligation
*adapters for ligation to Hind III ends are made by annealing oligos ADAPS-E1(5’-aattcacctgcccgg/3AmMc7/-3’) w/ a 3’ amino terminal end and ADAPL-E1(5’-ctaatacgactcactatagggctcgagcggccgcccgggcaggtg-3’). Oligos may be purchased from IDT at www.idtdna.com
-dilute ADAPS and ADAPL to 100uM
-combine in equal amts of ADAPS and ADAPL (i.e. add 10 µL of ADAPS add 10 uL of ADAPL)
-vortex briefly
-place tube in 500 mL of boiling H2O for 2 min
-remove heat and let bath cool for 1 hr. (this is to ensure correct nucleotide pairing)
-store adapter at -20C
5.) Construction of Adapter Library (ligate adapter to digestion)
-mix together:
10 uL cleaned gDNA digestion
1 uL Adapter (100uM)
2 uL T4 ligase (NEB product)
2 uL 10 x T4 ligase buffer (NEB buffer)
5 uL dH2O
TOTAL volume 20 uL
-vortex and incubate at 16C overnight in thermocycler
-heat inactivate at 65 for 20 min
-add 180 uL of TE (this is your adapter library store at -20C)
6.) Primers for 1º and 2º PCR
*Primary products are generated from amplifying primers AP1 (5’-ggatcctaatacgactcactataggc-3’) and PgwLat52LB-WP1 (5’-ctatgttactagatcgaccgg-3’).
*Secondary products are generated by diluting primary products by 50 fold and amplifying with primers AP2 (5’-tatagggctcgagcggccg-3’) and PgwLat52LB-WP2 (5’-caattcggcgttaattcagtac-3’).
-Primers come from IDT and must be diluted to 10 uM concentration before using.
7.) Primary PCR
-mix together:
0.125 uL Ex Taq (Takara)
2.5 uL 10 x Ex Taq Buffer
2.0 uL dNTP Mix
1.0 uL AP1
1.0 uL WP1
17.375 uL dH20
1.0 uL Adapter Library
TOTAL volume 25 µL
*Run on LMS_PCR2 (conditions recommended by Takara)
1.) incubate at 94C for 2 min
2.) incubate at 94C for 30 sec
3.) incubate at 55C for 30 sec
4.) incubate at 72C for 1 min
5.) recycle to step 2 for 29 more times
6.) incubate at 65C for 10 min
7.) hold at 4C forever
8.) Secondary PCR
*Dilute primary PCR:
-98 uL of dH20
-2 uL of primary PCR
*Rxn is setup exactly like primary PCR.
9.) Run on 1% Agarose Gel
5g Agarose Low
500mL 1 x TAE
50uL EtBr
*Always run a ladder to verify our bands are between 200 and 2000bp. Should only sequence the lines that give a clear band.
10.)Sequencing
*Need to clean up PCR rxn using Qiagen or Eppendorf kits. Then setup the rxn to be sequenced.
- mix together:
2 uL cleaned secondary PCR
1 uL secondary primer (WP2)
17 uL dH20
TOTAL volume 20 uL
*Sequencing is done through UNC Lineberger Comprehensive Cancer Center (located on campus), and normal turn around time can range from 1-7days.