Saturday, June 9, 2012

Cleaved Amplified Polymorphic Sequences (CAPS)

Cleaved Amplified Polymorphic Sequences (CAPS) polymorphisms are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites in PCR amplicons produced by locus-specific oligonucleotide primers.
How It Works
The CAPS assay uses amplified DNA fragments that are digested with a restriction endonuclease to display RFLP.

CAPS assay principle
Unique sequence primers are used to amplify a mapped DNA sequence from two related individuals (for example, from two different inbred ecotypes), A/A and B/B, and from the heterozygote A/B. The amplified fragments from A/A and B/B contain two and three RE recognition sites, respectively. In the case of the heterozygote A/B, two different PCR products will be obtained, one which is cleaved three times and one which is cleaved twice. When fractionated by agarose or acrylamide gel electrophoresis, the PCR products digested by the RE will give readily distinguishable patterns. Some bands will appear as doublets.
  • Most CAPS markers are co-dominant and locus-specific.
  • Most CAPS genotypes are easily scored and interpreted.
  • CAPS markers are easily shared between laboratories.
  • CAPS assay does not require the use of radioactive isotopes, and it is more amenable, therefore, to analyses in clinical settings.
Developing CAPS markers
  • Sequence the RFLP probe.
  • Design primers to amplify 800–2,000-bp DNA fragments. Targeting introns or 3' untranslated regions should increase the chance of finding polymorphisms
  • The PCR product is cloned and sequenced.
  • PCR amplify DNA fragments from target genotypes, separately digest the amplicons with one or more restriction emzymes.
  • Screen the digested amplicons for polymorphism on gels stained with ethidium bromide. 

The Derived Cleaved Amplified Polymorphic Sequences (dCAPS) assay is a modification of CAPS (or alternatively, PCR-RFLP) technique for detection of Single Nucleotide Polymorphisms (SNPs). In dCAPS assay a mismatches in PCR primer are used to create restriction endonuclease (RE)-sensitive polymorphism based on the target mutation. This technique is useful for genotyping known mutations and genetic mapping of isolated DNAs.
Similar to the CAPS technique, this method is simple, relatively inexpensive, and uses the ubiquitous technologies of PCR, restriction digestion and standard agarose gel electrophoresis.

How It Works
The dCAPS technique introduces or destroys a restriction enzyme recognition sites by using primers that containing one or more micmatches to the template DNA. The PCR product modified in this manner is then subjected to restriction enzyme digestion and the presence or absence of the SNP is determined by the resulting restriction pattern.

dCAPS assay example

Applications of dCAPS primers
1.        To create a restriciton site that is dependent on the presence or absence of the SNP allele in question
2.        To introduce a specific restriction site for each of two alleles being analyzed, to positively identify homozygotes for a particular allele without the possibbility of mis-scoring due to partial restriciton enzyme digestion
3.        To disrupt an additional restriction site situated in close proximity to the CAPS polymorphism to be analyzed

Amplified Fragment Length Polymorphism (AFLP)

Amplified Fragment Length Polymorphisms (AFLPs) are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites.

The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA.

AFLP principle
After final amplification, selectively amplified fragments are separated by gel electrophoresis and visualized autoradiographically. MseI-MseI fragments are excluded from the autorad because only EcoRI-directed primers are normally labeled. Typically, the autorad has 100-300 fingerprints with sizes ranging from 80 to 500 nucleotides. Only a subset (10-40) of these total bands is polymorphic between two related individuals, such as Arabidopsis thaliana Columbia and Landsberg erecta ecotypes.
Using 3-bp selective primer extensions gives 128 possible linker combinations. Therefore, 128 subsets of genomic DNA can be readily amplified. Thus, thousands of markers can be generated quite rapidly.

Weaknesses of AFLP
l        Proprietary technology is needed to score heterozygotes and ++ homozygotes. Otherwise, AFLP must be dominantly scored.
l        Developing locus-specific markers from individual fragments can be difficult.
l        Need to use different kits adapted to the size of the genome being analyzed.

Restriction Fragment Length Polymorphism (RFLP)

Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination.

Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific.

An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes.

The RFLP probes are frequently used in genome mapping and in variation analysis (genotyping, forensics, paternity tests, hereditary disease diagnostics, etc.).

How It Works
Principle of RFLP analysis
SNPs or INDELs can create or abolish restriction endonuclease (RE) recognition sites, thus affecting quantities and length of DNA fragments resulting from RE digestion.
RFLP genotyping

Developing RFLP probes
Total DNA is digested with a methylation-sensitive enzyme (for example, PstI), thereby enriching the library for single- or low-copy expressed sequences (PstI clones are based on the suggestion that expressed genes are not methylated).
The digested DNA is size-fractionated on a preparative agarose gel, and fragments ranging from 500 to 2000 bp are excised, eluted and cloned into a plasmid vector (for example, pUC18).
Digests of the plasmids are screened to check for inserts.
Southern blots of the inserts can be probed with total sheared DNA to select clones that hybridize to single- and low-copy sequences.
The probes are screened for RFLPs using genomic DNA of different genotypes digested with restriction endonucleases. Typically, in species with moderate to high polymorphism rates, two to four restriction endonucleases are used such as EcoRI
, EcoRV, and HindIII. In species with low polymorphism rates, additional restriction endonucleases can be tested to increase the chance of finding polymorphism.
Isolation of sufficient DNA for RFLP analysis is time consuming and labor intensive. However, PCR can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be analyzed in a shorter time. An alternative name for the technique is Cleaved Amplified Polymorphic Sequence (CAPS) assay.

Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA (RAPD) markers are DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence.

How It Works
Unlike traditional PCR analysis, RAPD (pronounced "rapid") does not require any specific knowledge of the DNA sequence of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers' sequence. For example, no fragment is produced if primers annealed too far apart or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in the template DNA at the site that was previously complementary to the primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel.

RAPD is an inexpensive yet powerful typing method for many bacterial species.

RAPD profiles, example

Silver-stained polyacrylamide gel showing three distinct RAPD profiles generated by primer OPE15 for Haemophilus ducreyi isolates from TanzaniaSenegalThailand, Europe, and North America.
Selecting the right sequence for the primer is very important because different sequences will produce different band patterns and possibly allow for a more specific recognition of individual strains.

Limitations of RAPD
Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). Co-dominant RAPD markers, observed as different-sized DNA segments amplified from the same locus, are detected only rarely.

PCR is an enzymatic reaction, therefore the quality and concentration of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully developed laboratory protocols to be reproducible.

Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.
Developing Locus-specific, Co-Dominant Markers from RAPDs
The polymorphic RAPD marker band is isolated from the gel.
It is amplified in the PCR reaction.
The PCR product is cloned and sequenced.
New longer and specific primers are designed for the DNA sequence, which is called the Sequenced Characterized Amplified Region Marker (SCAR).

Real-Time qRT-PCR

Real-Time qRT-PCR (Real Time quantitative Reverse Transcription PCR) is a major development of PCR technology that enables reliable detection and measurement of products generated during each cycle of PCR process. This technique became possible after introduction of an oligonucleotide probe, which was designed to hybridize within the target sequence. Cleavage of the probe during PCR because of the 5' nuclease activity of Taq polymerase can be used to detect amplification of the target-specific product.
How It Works
Principle of PCR
The following techniques can be used to monitor degradation of the probe:
  • intercalation of double-stranded DNA-binding dyes
  • 32P probe labeling
  • labeling of the probe with fluorescent dyes

TaqMan assay (named after Taq DNA polymerase) was one of the earliest methods introduced for real time PCR reaction monitoring and has been widely adopted for both the quantification of mRNAs and for detecting variation. The method exploits the 5' endonuclease activity of Taq DNA polymerase to cleave an oligonucleotide probe during PCR, thereby generating a detectable signal. The probes are fluorescently labeled at their 5' end and are non-extendable at their 3' end by chemical modification. Specificity is conferred at three levels: via two PCR primers and the probe. Applied Biosystems probes also include a minor groove binder for added specificity.

Applications of real time quantitative RT-PCR: relative and absolute quantification of gene expression, validation of DNA microarray results, variation analysis, counting bacterial, viral, or fungal loads, etc.
Model PCR plot
Nomenclature commonly used in real time quantitative RT-PCR:
Baseline is defined as PCR cycles in which a reporter fluorescent signal is accumulating but is beneath the limits of detection of the instrument.
ΔRn is an increment of fluorescent signal at each time point. The ΔRn values are plotted versus the cycle number.
Threshold is an arbitrary level of fluorescence chosen on the basis of the baseline variability. A signal that is detected above the threshold is considered a real signal that can be used to define the threshold cycle (Ct) for a sample. Threshold can be adjusted for each experiment so that it is in the region of exponential amplification across all plots.
Ct is defined as the fractional PCR cycle number at which the reporter fluorescence is greater than the threshold. The Ct is a basic principle of real time PCR and is an essential component in producing accurate and reproducible data.

Polymerase Chain Reaction (PCR) Illustrations

Polymerase chain reaction (PCR) is a laboratory technique used to amplify DNA sequences. The method involves using short DNA sequences called primers to select the portion of the genome to be amplified. The temperature of the sample is repeatedly raised and lowered to help a DNA replication enzyme copy the target DNA sequence. The technique can produce a billion copies of the target sequence in just a few hours.