Polymerase Chain Reaction (Molecular Info)

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

PCR (from Google directory)

A Rapid DNA Minipreparation Method Suitable for AFLP and Other PCR Applications - http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb17/17053-1.pdf 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 - http://info.med.yale.edu/genetics/ward/tavi/p16.html Brief discussion of additives to improve amplification efficiency and specificity of PCR, by Octavian Henegariu, Yale-New Haven Medical Center.

Amberg Laboratory Protocols: - http://www.upstate.edu/biochem/amberg/protocols.php Laboratory protocols for PCR work used by the group of David Amberg at the Department of Biochemistry and Molecular Biology Upstate Medical University, Syracuse, New York.

Anchor Probes for Comparative Mapping of Grass Species - http://greengenes.cit.cornell.edu/anchors/ 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 - http://www.attotron.com Research and development company for development of biosensors and related products for the research and educational markets.

BioRad, Amplification, PCR - http://www.bio-rad.com/B2B/BioRad/product/br_category.jsp? Division of BioRad Laboratories that manufactures and sells instruments for PCR, in Hercules, California, USA.

Degenerate PCR - http://www.dartmouth.edu/~ambros/protocols/other/koelle/degenerate_PCR.html 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.

Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences - http://www.uni-graz.at/~binder/thesis/node64.html Thesis abstract about restriction fragment length polymorphism (RFLP) by Alexander Binder 1997.

Detection of Single Nucleotide Mutations in Wheat Using Single Strand Conformation Polymorphism Gels - http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb19/R01-013.pdf P Martins-Lopez, H Zhang, R Koebner, Plant Mol. Biol. Reporter 19(2001): 159-162. From National Research Coouncil Canada.

DNALC: PCR Animation - http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html 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 - http://www.dnalc.org/home.html Educational site on topics in genetics and gene expression from Cold Spring Harbor Laboratory, USA.

Effect of PCR Buffer on Multiplex PCR - http://www.qiagen.com/literature/brochures/pcr/pdf/pcrcha03.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 - http://www.lecb.ncifcrf.gov/~pnh/papers/TIBS/aug95.html Article by PN Hengen from TIBS 1995

FISH Guide and Troubleshooting - http://info.med.yale.edu/genetics/ward/tavi/FISHguide.html 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.

Fluorescence in Situ Hybridisation - http://info.med.yale.edu/genetics/ward/tavi/FISH.html Technical notes on Fluorescence in situ hybridisation from the Institute of Genetics of Yale School of Medicine, New Haven, Connecticut, USA.

GeneOhm Sciences - http://www.geneohm.com Tests on group B Streptococcus and methicillin resistant Staphylococcus aureus by PCR / DNA sequencing.

GenHunter - http://www.genhunter.com Manufacturer of material for differential display PCR in Nashville, Tenn USA.

HiFi DNA - http://www.hifidna.com/ 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 - http://www.ingenetix.com/ 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 - http://www.fruitfly.org/about/methods/inverse.pcr.html Step by step protocol, by EJ Rehm, Berkeley Drosophila Genome Project, USA.

Inverse PCR for PAC-end Sequencing - http://www.genetics.wustl.edu/fish_lab/frank/cgi-bin/fish/prot2.html 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 - http://labs.fhcrc.org/gottschling/General%20Protocols/ipcr.html Protocol by M McMurray, Fred Hutchinson Cancer Research Center, Seattle, Wa. USA.

Inverse PCR Protocol - http://www.mcdb.lsa.umich.edu/labs/maddock/protocols/PCR/inverse_pcr_protocol.html Step by step protocol, from the web site of the Department of Biology, University of Michigan, USA.

Kary B. Mullis - Autobiography - http://nobelprize.org/chemistry/laureates/1993/mullis-autobio.html The originator of PCR, from the Nobel e-museum web site.

Kary Mullis - http://www.invent.org/hall_of_fame/109.html Inventor Profile of Kary Mullis, the originator of PCR, from the National Inventors Hall of Fame web site.

Long PCR Protocol - http://twod.med.harvard.edu/labgc/estep/longPCR_protocol.html 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 - http://nematode.unl.edu/its_id/EXAMPLES/index.htm Examples of Restriction Fragment Length Polymorphism (RFLP)electrophoresis slabs for different nematodes, from University of Nebrasca.

Optimizing Multiplex and LA-PCR with Betaine - http://www-lecb.ncifcrf.gov/~pnh/papers/TIBS/jun97.html LA-PCR = "long and accurate PCR". Article by PN Hengen in TIBS June 1997.

Optimizing PCR Protocols - http://www.jax.org/imr/optimize_pcr.html Brief guidelines. From the Jackson Laboratory, University of Maine, USA.

PCR Amplification of cDNA Segments by 2 Stage Nested PCR - http://www.ncbi.nlm.nih.gov/SNP/snp_viewTable.cgi?type=method&method_id=555 Protocol from the method database of NIH, USA.

PCR and Multiplex PCR Guide - http://www.info.med.yale.edu/genetics/ward/tavi/Guide.html 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.

PCR Animated - http://users.ugent.be/~avierstr/principles/pcrani.html Animation illustrating the principle of PCR, from the University of Ghent, Belgium.

PCR Gateway - http://www.horizonpress.com/pcr/ A directory of PCR techniques, PCR protocols, PCR troubleshooting, PCR websites and online resources from the publisher Horizon Press.

PCR Guru - http://www.pcrguru.com/ A downloadable textbook on PCR setup and optimization, not free.

PCR Method Protocols - http://hg.wustl.edu/hdk_lab_manual/pcr/pcrcontents.html Protocols for PCR posted by the Helen Donis-Keller Laboratory.

PCR Primer Design and Reaction Optimisation - http://web.uct.ac.za/microbiology/pcroptim.htm 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 - http://sunsite.berkeley.edu/biotech/pcr/ Presentations from the University of California at Berkley on PCR, both current research reports and reviews.

PCR Protocol - http://www.mcdb.lsa.umich.edu/labs/maddock/protocols/PCR/general_pcr_protocol.html Detailed PCR protocol from the web site of the Department of Biology, University of Michigan, USA.

PCR Protocols - http://www.cas.psu.edu/docs/CASDEPT/VET/jackvh/jvhpcr.html 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 - http://www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.html An introduction by Connie Veilleux from the US National Health Museum website.

PCR Troubleshooting - http://info.med.yale.edu/genetics/ward/tavi/Trblesht.html 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 World - http://pcrworld.blogspot.com Collection of unreferenced texts on various aspects of PCR.

PCR-ELISA and Related - http://www.btc-bti.com/pcrelisa.htm 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 - http://www.accessexcellence.com/RC/CT/polymerase_chain_reaction.html 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) - http://www.accessexcellence.org/AB/GG/polymerase.html A graphic description of the principle of PCR from the US National Health Museum web site.

PrimerDigital - http://primerdigital.com/index.php International biotechnology company specialized in a design service for PCR primers and probes, PCR-based technology development, projects for development of polymorphism and software development.

Primerfox - http://www.primerfox.com Free online tool for generation of PCR primers.

Principle of PCR - http://users.ugent.be/~avierstr/principles/pcr.html 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 - http://www.protocol-online.org/prot/Molecular_Biology/PCR/ Extensive collection of PCR protocols and methods from Protocol On Line.

Quantitative PCR Protocol - http://www.jax.org/cyto/quanpcr.html From the Jackson Laboratory, University of Maine, USA.

RAPD PCR - http://avery.rutgers.edu/WSSP/StudentScholars/project/archives/onions/rapd.html RAPD stands for Random Amplification of Polymorphic DNA, where the target sequence(s) (to be amplified) is unknown.Brief description, from Rutgers University, USA.

Rational Primer Design Greatly Improves Differential Display-PCR (DD-PCR) - http://nar.oxfordjournals.org/cgi/content/full/25/11/2239 Article: D Graf, AG Fisher, M Merkenschlager: Nucl. Acids Res. 25:11 2239-2240.

Reference in PCR - http://www.gene-quantification.org Technical aspects of quantitative real-time PCR and RT-PCR. Instruments, kits, dyes, chemistries, and services presented by their manufacturers.

Rep-PCR Genomic Fingerprinting - http://www.msu.edu/user/debruijn/ Bacteria are characterized by Rep-PCR fingerprinting using primers corresponding to naturally occurring repetitive sequences in the interspersed regions.

RFLP Definition - http://vm.cfsan.fda.gov/~frf/rflp.html RFLP = Restriction Fragment Length Polymorphism, from FDA

Roe Laboratory Protocols - http://www.genome.ou.edu/proto.html 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 - http://www-sequence.stanford.edu/group/yeast_deletion_project/single_tube_protocol.html 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 - http://web.uct.ac.za/microbiology/pcrcond.htm From Molecular Biology Techniques Manual, from the web site of the University of Cape Town, South Africa.

T-DNA Generated Enhancer Traps in Arabidopsis - http://www.dartmouth.edu/~tjack/ Application of inverse PCR, partial genomic libraries and TAIL-PCR in cloning flanking, at the Department of Biological Sciences, Dartmouth College, Hanover, NH.

Tavi's PCR Protocols - http://info.med.yale.edu/genetics/ward/tavi/PCR.html A page describing the main parameters and trouble-shooting in PCR. The page is somewhat dated (updated 1997) but still useful.

The PCR Encyclopedia - http://www.pcr-encyclopedia.com/ Describes plans for a free encyclopedia dedicated to the polymerase chain reaction (PCR).

The Web Guide of PCR - http://www.pcrlinks.com/ 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 - http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/hotpolys.html Discussion of their origin and briefly their properties. From the web site of Colorado State University.

Wayward PCR Primers - http://www-lecb.ncifcrf.gov/~pnh/papers/TIBS/jan95.html Article by PN Hengen from TIBS 1995 on the loss of activity of PCR primers with time.

What the Heck is PCR? - http://people.ku.edu/~jbrown/pcr.html Popular description of the PCR technique by John C Brown, University of Kansas 1995.

Which DNA Marker for Which Purpose? - http://webdoc.sub.gwdg.de/ebook/y/1999/whichmarker 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.

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].

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

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

Videos and Animations for PCR

YouTube - BC on Autism 17: A Primer on PCR
YouTube - PCR
YouTube - The qPCR le film FR
Fast PCR Tutorial
PCR reaction
Direct download: PCR movie (1.1 MB)
Direct download: PCR movie (800 KB)
PCR Animation
LinkedIn-PCR Tutorial
PCR--Introduction of 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

PCR Primer Design Tools

Primer3
PrimerQuest
Primer Premier
FastPCR
PrimerX
OligoMaster
PerlPrimer
Methprimer
NetPrimer
Oligo2002
CODEHOP
The Primer Generator
Primer Design Assistant
PROBEmer
GenomePride
Pride
TGGE-Star
Primer3 (UMass server)
Exon Locator and Extractor for Resequencing
AutoPrime -primer design software