Editing Polymerase chain reaction (section)

==Variations==
{{Main|Variants of PCR}}
* ''Allele-specific PCR'' or ''The amplification refractory mutation system (ARMS)'': a diagnostic or cloning technique based on single-nucleotide variations (SNVs not to be confused with [[Single-nucleotide polymorphism|SNPs]]) (single-base differences in a patient). Any mutation involving single base change can be detected by this system. It requires prior knowledge of a DNA sequence, including differences between [[allele]]s, and uses primers whose 3' ends encompass the SNV (base pair buffer around SNV usually incorporated).<ref>{{cite journal | vauthors = Bulduk, BK et al.| title = A Novel Amplification-Refractory Mutation System-PCR Strategy to Screen MT-TL1 Pathogenic Variants in Patient Repositories | journal = Genet Test Mol Biomarkers | volume = 24 | issue = 3 | pages = 165–170 | date = March 2020 | pmid = 32167396 | doi = 10.1089/gtmb.2019.0079 | s2cid = 212693790 }}</ref>  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 or small deletions in a sequence.<ref>{{cite journal | vauthors = Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF | title = Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS) | journal = Nucleic Acids Research | volume = 17 | issue = 7 | pages = 2503–16 | date = April 1989 | pmid = 2785681 | pmc = 317639 | doi = 10.1093/nar/17.7.2503 }}</ref> See [[SNP genotyping]] for more information.
* ''[[Polymerase cycling assembly|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.<ref name="Stemmer et al.">{{cite journal | vauthors = Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL | title = Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides | journal = Gene | volume = 164 | issue = 1 | pages = 49–53 | date = October 1995 | pmid = 7590320 | doi = 10.1016/0378-1119(95)00511-4 }}</ref>
* ''[[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.<ref name="Innis et al.">{{cite journal | vauthors = Innis MA, Myambo KB, Gelfand DH, Brow MA | title = DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 85 | issue = 24 | pages = 9436–40 | date = December 1988 | pmid = 3200828 | pmc = 282767 | doi = 10.1073/pnas.85.24.9436 | bibcode = 1988PNAS...85.9436I | doi-access = free }}</ref> A recent modification on this process, known as ''L''inear-''A''fter-''T''he-''E''xponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature ([[DNA melting|T<sub>m</sub>]]) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.<ref name="Pierce and Wangh">{{Cite book|vauthors=Pierce KE, Wangh LJ|title= Single Cell Diagnostics|chapter= Linear-After-The-Exponential Polymerase Chain Reaction and Allied Technologies|year= 2007|volume=132 |pages=65–85 |pmid=17876077 |doi=10.1007/978-1-59745-298-4_7 |series=Methods in Molecular Medicine|isbn=978-1-58829-578-1}}</ref>
* ''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.<ref>{{cite journal | vauthors = Krishnan M, Ugaz VM, Burns MA | title = PCR in a Rayleigh-Bénard convection cell | journal = Science | volume = 298 | issue = 5594 | pages = 793 | date = October 2002 | pmid = 12399582 | doi = 10.1126/science.298.5594.793 | author-link1 = Madhavi Krishnan }}</ref> 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.<ref>{{cite journal | vauthors = Priye A, Hassan YA, Ugaz VM | title = Microscale chaotic advection enables robust convective DNA replication | journal = Analytical Chemistry | volume = 85 | issue = 21 | pages = 10536–41 | date = November 2013 | pmid = 24083802 | doi = 10.1021/ac402611s }}</ref> Such convective flow PCR setup significantly reduces device power requirement and operation time.
* ''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.<ref name="Schwartz et al.">{{cite journal | vauthors = Schwartz JJ, Lee C, Shendure J | title = Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA molecules | journal = Nature Methods | volume = 9 | issue = 9 | pages = 913–15 | date = September 2012 | pmid = 22886093 | pmc = 3433648 | doi = 10.1038/nmeth.2137 }}</ref>
* ''[[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'.
* ''[[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.<ref>{{cite journal | vauthors = Vincent M, Xu Y, Kong H | title = Helicase-dependent isothermal DNA amplification | journal = EMBO Reports | volume = 5 | issue = 8 | pages = 795–800 | date = August 2004 | pmid = 15247927 | pmc = 1249482 | doi = 10.1038/sj.embor.7400200 }}</ref>
* ''[[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&nbsp;°C) before adding the polymerase.<ref name=general_hot_start>{{cite journal | vauthors = Chou Q, Russell M, Birch DE, Raymond J, Bloch W | title = Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications | journal = Nucleic Acids Research | volume = 20 | issue = 7 | pages = 1717–23 | date = April 1992 | pmid = 1579465 | pmc = 312262 | doi = 10.1093/nar/20.7.1717 }}</ref> Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an [[antibody]]<ref name=antibody_hot_start /><ref name="antibody_hot_start_2">{{cite journal | vauthors = Kellogg DE, Rybalkin I, Chen S, Mukhamedova N, Vlasik T, Siebert PD, Chenchik A | title = TaqStart Antibody: "hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase | journal = BioTechniques | volume = 16 | issue = 6 | pages = 1134–37 | date = June 1994 | pmid = 8074881 }}</ref> 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.
* ''[[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 [[Primer (molecular biology)|primers]] ([[Hybridization probe|probes]]) to amplify [[DNA]] sequences from a sequenced [[genome]] or [[transcriptome]]. In silico PCR was proposed as an educational tool for molecular biology.<ref>{{cite journal | vauthors = San Millán RM, Martínez-Ballesteros I, Rementeria A, Garaizar J, Bikandi J | title = Online exercise for the design and simulation of PCR and PCR-RFLP experiments | journal = BMC Research Notes | volume = 6 | pages = 513 | date = December 2013 | pmid = 24314313 | pmc = 4029544 | doi = 10.1186/1756-0500-6-513 | doi-access = free }}</ref>
* ''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.<ref>{{cite journal | vauthors = Zietkiewicz E, Rafalski A, Labuda D | title = Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification | journal = Genomics | volume = 20 | issue = 2 | pages = 176–83 | date = March 1994 | pmid = 8020964 | doi = 10.1006/geno.1994.1151 | s2cid = 41802285 }}</ref>
* ''[[Inverse polymerase chain reaction|Inverse PCR]]'': is commonly used to identify the flanking sequences around [[genomic]] inserts. It involves a series of [[Restriction digest|DNA digestions]] and [[self ligation]], resulting in known sequences at either end of the unknown sequence.<ref>{{cite journal | vauthors = Ochman H, Gerber AS, Hartl DL | title = Genetic applications of an inverse polymerase chain reaction | journal = Genetics | volume = 120 | issue = 3 | pages = 621–23 | date = November 1988 | doi = 10.1093/genetics/120.3.621 | pmid = 2852134 | pmc = 1203539 }}</ref>
* ''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]].<ref name="Mueller and Wold">{{cite journal | vauthors = Mueller PR, Wold B | title = In vivo footprinting of a muscle specific enhancer by ligation mediated PCR | journal = Science | volume = 246 | issue = 4931 | pages = 780–86 | date = November 1989 | pmid = 2814500 | doi = 10.1126/science.2814500 | bibcode = 1989Sci...246..780M }}</ref>
* ''[[Methylation-specific PCR]]'' (MSP): developed by [[Stephen Baylin]] and [[James G. Herman]] at the Johns Hopkins School of Medicine,<ref>{{cite journal | vauthors = Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB | title = Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 93 | issue = 18 | pages = 9821–26 | date = September 1996 | pmid = 8790415 | pmc = 38513 | doi = 10.1073/pnas.93.18.9821 | author-link1 = James G. Herman | bibcode = 1996PNAS...93.9821H | doi-access = free }}</ref> and 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.
* ''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.<ref>{{cite journal | vauthors = Isenbarger TA, Finney M, Ríos-Velázquez C, Handelsman J, Ruvkun G | title = Miniprimer PCR, a new lens for viewing the microbial world | journal = Applied and Environmental Microbiology | volume = 74 | issue = 3 | pages = 840–49 | date = February 2008 | pmid = 18083877 | pmc = 2227730 | doi = 10.1128/AEM.01933-07 | bibcode = 2008ApEnM..74..840I }}</ref>
* ''[[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).
* ''[[Multiplex-PCR]]'': consists of multiple primer sets within a single PCR mixture to produce [[amplicon]]s of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes. That is, their base pair length should be different enough to form distinct bands when visualized by [[gel electrophoresis]].
* ''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.<ref>{{cite journal | vauthors = Shen C, Yang W, Ji Q, Maki H, Dong A, Zhang Z | title = NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions | journal = Nanotechnology | volume = 20 | issue = 45 | pages = 455103 | date = November 2009 | pmid = 19822925 | doi = 10.1088/0957-4484/20/45/455103 | s2cid = 3393115 | bibcode = 2009Nanot..20S5103S }}</ref><ref>{{cite book|last=Shen|first=Cenchao|title=Bio-Nanotechnology |chapter=An Overview of Nanoparticle-Assisted Polymerase Chain Reaction Technology|year=2013|publisher=Wiley-Blackwell Publishing Ltd.|place=US|pages=97–106|doi=10.1002/9781118451915.ch5|isbn=978-1-118-45191-5}}</ref>
* ''[[Nested PCR]]'': increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
* ''[[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.<ref>{{cite journal | vauthors = Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR | title = Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension | journal = Gene | volume = 77 | issue = 1 | pages = 61–68 | date = April 1989 | pmid = 2744488 | doi = 10.1016/0378-1119(89)90359-4 }}</ref><ref>{{cite book|last=Moller|first=Simon|title=PCR: The Basics|isbn=978-0-415-35547-6|year=2006|publisher=Taylor & Francis Group|location=US|page=144}}</ref>
* ''PAN-AC'': uses isothermal conditions for amplification, and may be used in living cells.<ref name="DavidTurlotte1998">{{cite journal | vauthors = David F, Turlotte E | title = [A method of isothermal gene amplification] | journal = Comptes Rendus de l'Académie des Sciences, Série III | volume = 321 | issue = 11 | pages = 909–14 | date = November 1998 | pmid = 9879470 | doi = 10.1016/S0764-4469(99)80005-5 | trans-title = An Isothermal Amplification Method | bibcode = 1998CRASG.321..909D }}</ref><ref>{{cite web| url=http://www.lab-rech-associatives.com/pdf/Utiliser%20la%20Topologie%20de%20l'ADN.pdf| archive-url=https://web.archive.org/web/20071128140836/http://www.lab-rech-associatives.com/pdf/Utiliser%20la%20Topologie%20de%20l'ADN.pdf| url-status=usurped| archive-date=2007-11-28| title=Utiliser les propriétés topologiques de l'ADN: une nouvelle arme contre les agents pathogènes |publisher=Fusion| volume=92|date=September–October 2002|author=Fabrice David}}(in French)</ref>
* ''PAN-PCR'': A computational method for designing bacterium typing assays based on whole genome sequence data.<ref>{{cite journal | doi=10.1128/jcm.02671-12 | title=Pan-PCR, a Computational Method for Designing Bacterium-Typing Assays Based on Whole-Genome Sequence Data | date=2013 | last1=Yang | first1=Joy Y. | last2=Brooks | first2=Shelise | last3=Meyer | first3=Jennifer A. | last4=Blakesley | first4=Robert R. | last5=Zelazny | first5=Adrian M. | last6=Segre | first6=Julia A. | last7=Snitkin | first7=Evan S. | journal=Journal of Clinical Microbiology | volume=51 | issue=3 | pages=752–758 | pmid=23254127 | doi-access=free | pmc=3592046 }}</ref>
* ''[[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 [[Quantitative PCR|RT-PCR]] (''real-time'' PCR) but this abbreviation should be used only for [[Reverse transcription polymerase chain reaction|reverse transcription PCR]]. qPCR is the appropriate contractions for [[quantitative PCR]] (real-time PCR).
* ''[[Reverse complement PCR]]'' (RC-PCR): Allows the addition of functional domains or sequences of choice to be appended independently to either end of the generated amplicon in a single closed tube reaction. This method generates target specific primers within the reaction by the interaction of universal primers (which contain the desired sequences or domains to be appended) and RC probes.
* ''[[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 (biology)|RACE-PCR]] (''Rapid Amplification of cDNA Ends'').
* ''[[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.<ref name="dobosy_2011">{{cite journal | vauthors = Dobosy JR, Rose SD, Beltz KR, Rupp SM, Powers KM, Behlke MA, Walder JA | title = RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers | journal = BMC Biotechnology | volume = 11 | pages = 80 | date = August 2011 | pmid = 21831278 | pmc = 3224242 | doi = 10.1186/1472-6750-11-80 | doi-access = free }}</ref>
* {{anchor|SSP-PCR}}''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.<ref>{{Cite book|last1=Shyamala|first1=V.|last2=Ferro-Luzzi Ames|first2= G.|title=PCR Protocols |chapter=Single Specific Primer-Polymerase Chain Reaction (SSP-PCR) and Genome Walking |series=Methods in Molecular Biology|date=1993|volume=15|pages=339–48|doi=10.1385/0-89603-244-2:339|pmid=21400290|isbn=978-0-89603-244-6}}</ref>
* ''Solid phase PCR'': encompasses multiple meanings, including [[Polony (biology)|polony amplification]] (where PCR colonies are derived in a gel matrix, for example), bridge PCR<ref>{{cite journal|title=Bridge amplification: a solid phase PCR system for the amplification and detection of allelic differences in single copy genes|journal=Genetic Identity Conference Proceedings, Seventh International Symposium on Human Identification|year=1996|vauthors=Bing DH, Boles C, Rehman FN, Audeh M, Belmarsh M, Kelley B, Adams CP|url=http://www.promega.com/geneticidproc/ussymp7proc/0726.html|archive-url=https://web.archive.org/web/20010507195511/http://www.promega.com/geneticidproc/ussymp7proc/0726.html|url-status=dead|archive-date=7 May 2001}}</ref> (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<ref>{{cite journal | vauthors = Khan Z, Poetter K, Park DJ | title = Enhanced solid phase PCR: mechanisms to increase priming by solid support primers | journal = Analytical Biochemistry | volume = 375 | issue = 2 | pages = 391–93 | date = April 2008 | pmid = 18267099 | doi = 10.1016/j.ab.2008.01.021 }}</ref> (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).
* ''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 the plague during the medieval [[Black Death]] epidemic.<ref>{{cite journal | vauthors = Raoult D, Aboudharam G, Crubézy E, Larrouy G, Ludes B, Drancourt M | title = Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 97 | issue = 23 | pages = 12800–03 | date = November 2000 | pmid = 11058154 | pmc = 18844 | doi = 10.1073/pnas.220225197 | bibcode = 2000PNAS...9712800R | doi-access = free }}</ref> 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.
* ''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.<ref>{{cite journal | vauthors = Liu YG, Whittier RF | title = Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking | journal = Genomics | volume = 25 | issue = 3 | pages = 674–81 | date = February 1995 | pmid = 7759102 | doi = 10.1016/0888-7543(95)80010-J }}</ref>
* ''[[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&nbsp;°C) above the T<sub>m</sub> of the primers used, while at the later cycles, it is a few degrees (3–5&nbsp;°C) below the primer T<sub>m</sub>. 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.<ref>{{cite journal | vauthors = Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS | title = 'Touchdown' PCR to circumvent spurious priming during gene amplification | journal = Nucleic Acids Research | volume = 19 | issue = 14 | pages = 4008 | date = July 1991 | pmid = 1861999 | pmc = 328507 | doi = 10.1093/nar/19.14.4008 }}</ref>
* Two-Tailed PCR is a technology developed by Professor [[Mikael Kubista]] to amplify short template molecules like [[microRNA]]s and even shorter using a hairpin primer that hybridizes to the target with both its 3' and 5'-ends.<ref>{{Cite journal |last1=Androvic |first1=Peter |last2=Valihrach |first2=Lukas |last3=Elling |first3=Julie |last4=Sjoback |first4=Robert |last5=Kubista |first5=Mikael |date=2017-09-06 |title=Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification |url=https://academic.oup.com/nar/article/45/15/e144/3958703 |journal=Nucleic Acids Research |volume=45 |issue=15 |pages=e144 |doi=10.1093/nar/gkx588 |pmid=28911110 |pmc=5587787 |issn=0305-1048}}</ref> 
* ''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')<ref name="Myrick and Gelbart">{{cite journal | vauthors = Myrick KV, Gelbart WM | title = Universal Fast Walking for direct and versatile determination of flanking sequence | journal = Gene | volume = 284 | issue = 1–2 | pages = 125–31 | date = February 2002 | pmid = 11891053 | doi = 10.1016/S0378-1119(02)00384-0 }}</ref> by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends),<ref name="lane rage">{{Cite web|url=http://www.ejbiotechnology.info/content/vol8/issue2/full/7/index.html|title=Full Text – LaNe RAGE: a new tool for genomic DNA flanking sequence determination|website=www.ejbiotechnology.info|access-date=24 April 2008|archive-date=16 May 2008|archive-url=https://web.archive.org/web/20080516110839/http://www.ejbiotechnology.info/content/vol8/issue2/full/7/index.html|url-status=dead}}</ref> 5'RACE LaNe<ref name="Parkb">{{cite journal | vauthors = Park DJ | title = A new 5' terminal murine GAPDH exon identified using 5'RACE LaNe | journal = Molecular Biotechnology | volume = 29 | issue = 1 | pages = 39–46 | date = January 2005 | pmid = 15668518 | doi = 10.1385/MB:29:1:39 | s2cid = 45702164 }}</ref> and 3'RACE LaNe.<ref name="Park">{{cite journal | vauthors = Park DJ | title = 3' RACE LaNe: a simple and rapid fully nested PCR method to determine 3'-terminal cDNA sequence | journal = BioTechniques | volume = 36 | issue = 4 | pages = 586–88, 590 | date = April 2004 | pmid = 15088375 | doi = 10.2144/04364BM04 | doi-access = free }}</ref>