Variants of PCR

Variants of PCR represent a diverse array of techniques that have evolved from the basic polymerase chain reaction method, each tailored to specific applications in molecular biology, such as genetic analysis, DNA sequencing, and disease diagnosis, by modifying factors like primer design, temperature conditions, and enzyme usage.

Basic modifications

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. This permits the simultaneous analysis of multiple targets in a single sample. For example, in testing for genetic mutations, six or more amplifications might be combined. In the standard protocol for DNA fingerprinting, the targets assayed are often amplified in groups of 3 or 4. Multiplex Ligation-dependent Probe Amplification permits multiple targets to be amplified using only a single pair of primers, avoiding the resolution limitations of multiplex PCR. Multiplex PCR has also been used for analysis of microsatellites and SNPs.
Variable Number of Tandem Repeats PCR targets repetitive areas of the genome that exhibit length variation. Analysis of the genotypes in the samples usually involves sizing of the amplification products by gel electrophoresis. Analysis of smaller VNTR segments known as short tandem repeats is the basis for DNA fingerprinting databases such as CODIS.
Asymmetric PCR preferentially amplifies one strand of a double-stranded DNA target. It is used in some sequencing methods and hybridization probing to generate one DNA strand as product. Thermocycling is carried out exactly as in conventional PCR, but with a limiting amount or leaving out one of the primers. When the limiting primer becomes depleted, replication increases arithmetically rather than exponentially through extension of the excess primer. A modification of this process, named Linear-After-The-Exponential-PCR, uses a limiting primer with a higher melting temperature than the excess primer in order to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction. See also overlap-extension PCR.
Some modifications are needed to perform long PCR. The original Klenow-based PCR process did not generate products that were larger than about 400 bp. Taq polymerase can however amplify targets of up to several thousand bp long. Since then, modified protocols with Taq enzyme have allowed targets of over 50 kb to be amplified.
Nested PCR is used to increase the specificity of DNA amplification. Two sets of primers are used in two successive reactions. In the first PCR, one pair of primers is used to generate DNA products, which may contain products amplified from non-target areas. The products from the first PCR are then used as template in a second PCR, using one or two different primers whose binding sites are located within the first set, thus increasing specificity. 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 is used to measure the specific amount of target DNA in a sample. By measuring amplification only within the phase of true exponential increase, the amount of measured product more accurately reflects the initial amount of target. Special thermal cyclers are used that monitor the amount of product during the amplification.
Quantitative Real-Time PCR, sometimes simply called Real-Time 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 before adding the polymerase. In this way, non-specific amplification at lower temperatures is prevented. Alternatively, specialized reagents 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.
In touchdown PCR, the annealing temperature is gradually decreased in later cycles. The annealing temperature in the early cycles is usually 3–5 °C above the standard T_m of the primers used, while in the later cycles it is a similar amount below the T_m. The initial higher annealing temperature leads to greater specificity for primer binding, while the lower temperatures permit more efficient amplification at the end of the reaction.
Assembly PCR is the synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments, to assemble two or more pieces of DNA into one piece. It involves an initial PCR with primers that have an overlap and a second PCR using the products as the template that generates the final full-length product. This technique may substitute for ligation-based assembly.
In colony PCR, bacterial colonies are screened directly by PCR, for example, the screen for correct DNA vector constructs. Colonies are sampled with a sterile pipette tip and a small quantity of cells transferred into a PCR mix. To release the DNA from the cells, the PCR is either started with an extended time at 95 °C, or with a shortened denaturation step at 100 °C and special chimeric DNA polymerase.
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. The method prescribes the use of any primer combination only once in a PCR, which should never have been used in any positive-control PCR reaction, and the primers should always target a genomic region never amplified before in the lab using this or any other set of primers. This ensures that no contaminating DNA from previous PCR reactions is present in the lab, which could otherwise generate false positives.
COLD-PCR is a modified protocol that enriches variant alleles from a mixture of wild-type and mutation-containing DNA samples.

Pretreatments and extensions

The basic PCR process can sometimes precede or follow another technique.
RT-PCR is used to reverse-transcribe and amplify RNA to cDNA. 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. The Tth polymerase has RT activity, and can carry out the entire reaction. RT-PCR is widely used in expression profiling, which detects the expression of a gene. It can also be used to obtain sequence of an RNA transcript, which may aid the determination of the transcription start and termination sites and facilitate mapping of the location of exons and introns in a gene sequence. Multiplex qRT-PCR has been developed to enhance SARS-CoV-2 detection efficiency by allowing multiple viral targets to be tested simultaneously.
Two-tailed PCR uses a single primer that binds to a microRNA target with both 3' and 5' ends, known as hemiprobes. Both ends must be complementary for binding to occur. The 3'-end is then extended by reverse transcriptase forming a long cDNA. The cDNA is then amplified using two target specific PCR primers. The combination of two hemiprobes, both targeting the short microRNA target, makes the Two-tailed assay exceedingly sensitive and specific.
Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' that are first ligated to fragments of the target DNA. PCR primers that anneal to the linker sequences are then used to amplify the target fragments. This method is deployed for DNA sequencing, genome walking, and DNA footprinting. A related technique is amplified fragment length polymorphism, which generates diagnostic fragments of a genome.
Methylation-specific PCR is used to identify patterns of DNA methylation at cytosine-guanine islands in genomic DNA. Target DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is complementary to adenosine in PCR primers. Two amplifications are then carried out on the bisulfite-treated DNA: one primer set anneals to DNA with cytosines, and the other set anneals to DNA with uracil. MSP used in quantitative PCR provides quantitative information about the methylation state of a given CpG island.

Other modifications

Adjustments of the components in PCR is commonly used for optimal performance.
The divalent magnesium ion is required for PCR polymerase activity. Lower concentrations Mg⁺⁺ will increase replication fidelity, while higher concentrations will introduce more mutations.
Denaturants can increase amplification specificity by destabilizing non-specific primer binding. Other chemicals, such as glycerol, are stabilizers for the activity of the polymerase during amplification. Detergents can prevent polymerase stick to itself or to the walls of the reaction tube.
DNA polymerases occasionally incorporate mismatch bases into the extending strand. High-fidelity PCR employs enzymes with 3'-5' exonuclease activity that decreases this rate of mis-incorporation. 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.