Variants of PCR

1. Allele-specific PCR: a diagnostic or cloning technique based on single-nucleotide

variations (SNVs not to be confused with SNPs) (single-base differences in a patient). It
requires prior knowledge of a DNA sequence, including differences between alleles, and
uses primers whose 3' ends encompass the SNV (base pair buffer around SNV usually
incorporated). 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.
2. Assembly PCR or Polymerase Cycling Assembly (PCA): 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.
3. Asymmetric PCR: preferentially amplifies one DNA strand in a double-stranded DNA
template. It is used in sequencing and hybridization probing where amplification of only
one of the two complementary strands is required. PCR is carried out as usual, but with
a great excess of the primer for the strand targeted for amplification. Because of the
slow (arithmetic) amplification later in the reaction after the limiting primer has been
used up, extra cycles of PCR are required. A recent modification on this process, known
as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher
melting temperature (Tm) than the excess primer to maintain reaction efficiency as the
limiting primer concentration decreases mid-reaction.
4. Convective PCR: a pseudo-isothermal way of performing PCR. Instead of repeatedly
heating and cooling the PCR mixture, the solution is subjected to a thermal gradient.
The resulting thermal instability driven convective flow automatically shuffles the PCR
reagents from the hot and cold regions repeatedly enabling PCR. Parameters such as
thermal boundary conditions and geometry of the PCR enclosure can be optimized to
yield robust and rapid PCR by harnessing the emergence of chaotic flow fields. Such
convective flow PCR setup significantly reduces device power requirement and
operation time.
5. Dial-out PCR: a highly parallel method for retrieving accurate DNA molecules for gene
synthesis. A complex library of DNA molecules is modified with unique flanking tags
before massively parallel sequencing. Tag-directed primers then enable the retrieval of
molecules with desired sequences by PCR.
6. Digital PCR (dPCR): used to measure the quantity of a target DNA sequence in a DNA
sample. The DNA sample is highly diluted so that after running many PCRs in parallel,
some of them do not receive a single molecule of the target DNA. The target DNA
concentration is calculated using the proportion of negative outcomes. Hence the name
'digital PCR'.
7. Helicase-dependent amplification: 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.
8. Hot start PCR: a technique that reduces non-specific amplification during the initial set
up stages of the PCR. It may be performed manually by heating the reaction
components to the denaturation temperature (e.g., 95 °C) before adding the
polymerase. Specialized enzyme 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 dissociate only 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.
9. In silico PCR (digital PCR, virtual PCR, electronic PCR, e-PCR) refers to computational
tools used to calculate theoretical polymerase chain reaction results using a given set
of primers(probes) to amplify DNA sequences from a
sequenced genome or transcriptome. In silico PCR was proposed as an educational tool
for molecular biology.
10. Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies
regions between simple sequence repeats to produce a unique fingerprint of amplified
fragment lengths.
11. Inverse PCR: is commonly used to identify the flanking sequences
around genomic inserts. It involves a series of DNA digestions and self-ligation, resulting
in known sequences at either end of the unknown sequence.
12. Ligation-mediated PCR: 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.
13. Methylation-specific PCR (MSP): 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.
14. Miniprimer PCR: uses a thermostable polymerase (S-Tbr) that can extend from short
primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting
to smaller primer binding regions, and is used to amplify conserved DNA sequences,
such as the 16S (or eukaryotic 18S) rRNA gene.
15. Multiplex ligation-dependent probe amplification (MLPA): permits amplifying multiple
targets with a single primer pair, thus avoiding the resolution limitations of multiplex
PCR (see below).
16. Nanoparticle-Assisted PCR (nanoPCR): some nanoparticles (NPs) can enhance the
efficiency of PCR (thus being called nanoPCR), and some can even outperform the
original PCR enhancers. It was reported that quantum dots (QDs) can improve PCR
 specificity and efficiency. Single-walled carbon nanotubes (SWCNTs) and multi-walled
carbon nanotubes (MWCNTs) are efficient in enhancing the amplification of long PCR.
Carbon nanopowder (CNP) can improve the efficiency of repeated PCR and long PCR,
while zinc oxide, titanium dioxide and Ag NPs were found to increase the PCR yield.
Previous data indicated that non-metallic NPs retained acceptable amplification fidelity.
Given that many NPs are capable of enhancing PCR efficiency, it is clear that there is
likely to be great potential for nanoPCR technology improvements and product
development.
17. Overlap-extension PCR or Splicing by overlap extension (SOEing): a genetic
engineering technique that is used to splice together two or more DNA fragments that
contain complementary sequences. It is used to join DNA pieces containing genes,
regulatory sequences, or mutations; the technique enables creation of specific and long
DNA constructs. It can also introduce deletions, insertions or point mutations into a DNA
sequence.
18. PAN-AC: uses isothermal conditions for amplification, and may be used in living cells.
19. quantitative PCR (qPCR): used to measure the quantity of a target sequence (commonly
in real-time). It quantitatively measures starting amounts of DNA, cDNA, or
RNA. quantitative PCR is commonly used to determine whether a DNA sequence is
present in a sample and the number of its copies in the sample. Quantitative PCR has a
very high degree of precision. Quantitative PCR methods use fluorescent dyes, such as
Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to
measure the amount of amplified product in real time. It is also sometimes abbreviated
to RT-PCR (real-time PCR) but this abbreviation should be used only for reverse
transcription PCR. qPCR is the appropriate contractions for quantitative PCR (real-time
PCR).
20. Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse
transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. 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. If the genomic DNA sequence of a gene is known, RT-PCR can be used 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 RACE-PCR(Rapid Amplification of cDNA
Ends).
21. RNase H-dependent PCR (rhPCR): a modification of PCR that utilizes primers with a 3’
extension block that can be removed by a thermostable RNase HII enzyme. This system
reduces primer-dimers and allows for multiplexed reactions to be performed with
higher numbers of primers.
22. Single Specific Primer-PCR (SSP-PCR): allows the amplification of double-stranded DNA
even when the sequence information is available at one end only. This method permits
amplification of genes for which only a partial sequence information is available, and
allows unidirectional genome walking from known into unknown regions of the
chromosome.
23. Solid Phase PCR: encompasses multiple meanings, including Polony
Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge
PCR (primers are covalently linked to a 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
(where conventional Solid Phase PCR can be improved by employing high Tm and nested
solid support primer with optional application of a thermal 'step' to favour solid support
priming).
24. Suicide PCR: 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 (hence the term "suicide"), 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.
25. Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an 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.
26. Touchdown PCR (Step-down 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.
27. Universal Fast Walking: for 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') by virtue
of a mechanism involving lariat structure formation.