Variants of PCR

The versatility of polymerase chain reaction (PCR) has led to modifications of the basic protocol being used in a large number of variant techniques designed for various purposes.

This article summarizes many of the most common variations currently or formerly used in molecular biology laboratories; familiarity with the fundamental premise by which PCR works and corresponding terms and concepts is necessary for understanding these variant techniques.

Often only a small modification needs to be made to the standard PCR protocol to achieve a desired goal: Multiplex-PCR uses several pairs of primers annealing to different target sequences.

[1] Variable Number of Tandem Repeats (VNTR) PCR targets repetitive areas of the genome that exhibit length variation.

Analysis of smaller VNTR segments known as short tandem repeats (or STRs) is the basis for DNA fingerprinting databases such as CODIS.

Quantitative PCR (qPCR) is used to measure the specific amount of target DNA (or RNA) in a sample.

Quantitative Real-Time PCR (QRT-PCR), sometimes simply called Real-Time PCR (RT-PCR), refers to a collection of methods that 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 as the amplification progresses.

Hot-start PCR is a technique performed manually by heating the reaction components to the DNA melting temperature (e.g. 95 °C) before adding the polymerase.

Colonies are sampled with a sterile pipette tip and a small quantity of cells transferred into a PCR mix.

[9] The digital polymerase chain reaction simultaneously amplifies thousands of samples, each in a separate droplet within an emulsion or partition within an micro-well.

Suicide PCR is typically used in paleogenetics or other studies where avoiding false positives and ensuring the specificity of the amplified fragment is the highest priority.

It was originally described in a study to verify the presence of the microbe Yersinia pestis in dental samples obtained from 14th-century graves of people supposedly killed by plague during the medieval Black Death epidemic.

This ensures that no contaminating DNA from previous PCR reactions is present in the lab, which could otherwise generate false positives.

COLD-PCR (co-amplification at lower denaturation temperature-PCR) is a modified protocol that enriches variant alleles from a mixture of wild-type and mutation-containing DNA samples.

PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA.

The two reactions may be combined in a tube, with the initial heating step of PCR being used to inactivate the transcriptase.

[13] Target DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is complementary to adenosine in PCR primers.

Examples of enzymes with proofreading activity include Pfu; adjustments of the Mg++ and dNTP concentrations may help maximize the number of products that exactly match the original target DNA.

(also see Overlap extension polymerase chain reaction) Normally the primers used in PCR are designed to be fully complementary to the target.

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.

The method utilizes primers with a cleavable block on the 3’ end that is removed by the action of a thermostable RNase HII enzyme.

Tth has reverse transcriptase activity in the presence of Mn2+ ions, allowing PCR amplification from RNA targets.

[23] The archean genus Pyrococcus has proven a rich source of thermostable polymerases with proofreading activity.

Pfu DNA polymerase, isolated from the P. furiosus shows a 5-fold decrease in the error rate of replication compared to Taq.

[24] Since errors increase as PCR progresses, Pfu is the preferred polymerase when products are to be individually cloned for sequencing or expression.

It involves initially subjecting the target DNA to a series of restriction enzyme digestions, and then circularizing the resulting fragments by self ligation.

[26] Similarly, thermal asymmetric interlaced PCR (or TAIL-PCR) is used to isolate unknown sequences flanking a known area of the genome.

[28] Helicase-dependent amplification (HDA) is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension steps.

The reaction progresses rapidly and results in specific DNA amplification from just a few target copies to detectable levels typically within 5–10 minutes.