Polymerase Chain Reaction (PCR)


Polymerase chain reaction (PCR) is a rapid, in vitrodeoxyribonucleic acid (DNA) synthesis process, which can amplify up to a billion copies of a given nucleic acid target. It has been extensively applied for the identification, detection and diagnosis of genetic and infectious disease. In the article, we provide a general overview of the PCR process, describe methods to identify and minimize exogenous contamination, and highlight limitations of product size and polymerase‐dependent errors. We present many of the applications for which PCR is used, particularly in creating recombinant DNA constructs, in detecting genetic variation, in preparing templates for DNA sequence analysis and in quantitating gene transcripts or viral copies. Whole genome amplification and the ligation chain reaction are also presented, which we believe this article provides the reader with a comprehensive viewpoint of the variety of amplification‐based methods used in molecular biology today.

Key Concepts

  • Polymerase chain reaction is an in vitro amplification method widely used in molecular biology.

  • PCR contamination can occur from a single molecule of foreign or exogenous DNA, which, if gone unchecked, can confound the interpretation of results.

  • PCR cloning of full‐length genes can be problematic because mutations can result in amino acid substitutions in wild‐type sequences.

  • PCR can reliably amplify target sizes up to 3–4 kb from a variety of source materials.

  • Recombinant PCR products can be created by mismatching sequences between the primer and the template DNA or by adding 5′ exogenous sequences to the primers.

  • PCR can detect human genetic variation associated with hereditary disease and cancer.

  • Degenerate PCR is a powerful tool for identifying novel gene family members that are important in drug development.

  • Quantitative PCR has been widely used for studying gene expression and estimating viral copy number.

  • Emulsion PCR is used to prepare templates in a cell‐free system for next‐generation sequencing platforms.

  • Whole genome amplification uses the highly processive φ29 DNA polymerase to amplify nanogram quantities of precious samples into microgram amounts.

Keywords: polymerase chain reaction; PCR; Taq DNA polymerase; multiplex PCR; emulsion PCR; whole genome amplification

Figure 1.

The PCR amplification cycle.

Figure 2.

Creation of mutagenized or recombinant PCR products via primer mismatches (left) or 5′‐add‐on sequences (right).



Barany F (1991) Genetic disease detection and DNA amplification using cloned thermostable ligase. Proceedings of the National Academy of Sciences of the USA 88: 189–193.

Barnes WM (1994) PCR amplification of up to 35‐kb DNA with high fidelity and high yield from λ bacteriophage templates. Proceedings of the National Academy of Sciences of the USA 91: 2216–2220.

Chamberlain JS, Gibbs RA, Rainier JE, Nguyen PN and Caskey CT (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Research 23: 11141–11156.

Cheng S, Fockler C, Barnes WM and Higuchi R (1994) Effective amplification of long targets from cloned inserts and human genomic DNA. Proceedings of the National Academy of Sciences of the USA 91: 5695–5699.

Cline J, Braman JC and Hogrefe HH (1996) PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Research 24: 3546–3551.

Dalton R (1999) Roche's Taq patent ‘obtained by deceit’, rules US court. Nature 402: 709.

Dean FB, Hosono S, Fang L et al. (2002) Comprehensive human genome amplification using multiple displacement amplification. Proceedings of the National Academy of Sciences of the USA 99: 5261–5266.

Dressman D, Yan H, Traverso G, Kinzler KW and Vogelstein B (2003) Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proceedings of the National Academy of Sciences of the USA 100: 8817–8822.

Eckert KA and Kunkel TA (1990) High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Research 18: 3739–3744.

Gibbs RA and Chamberlain JS (1989) The polymerase chain reaction: a meeting report. Genes and Development 3: 1095–1098.

Gibbs RA, Nguyen PN, Edwards A, Civitello AB and Caskey CT (1990) Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in Lesch–Nyhan families. Genomics 7: 235–244.

Gibson UEM, Heid CA and Williams PM (1996) A novel method for real time quantitative RT‐PCR. Genome Research 6: 995–1001.

Gilliland G, Perrin S, Blanchard K and Bunn HF (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proceedings of the National Academy of Sciences of the USA 87: 2725–2729.

Golenberg EM, Gainnasi DE, Clegg MT et al. (1990) Chloroplast DNA sequence from a Miocene Magnolia species. Nature 344: 656–658.

Guatelli JC, Whitfield KM, Kwok DY et al. (1990) Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proceedings of the National Academy of Sciences of the USA 87: 1874–1878.

Heid CA, Stevens J, Livak KJ and Williams PM (1996) Real time quantitative PCR. Genome Research 6: 986–994.

Holland PM, Abramson RD, Watson R and Gelfand DH (1991) Detection of specific polymerase chain reaction product by utilizing the 5′→3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proceedings of the National Academy of Sciences of the USA 88: 7276–7280.

Hosono S, Faruqi FA, Dean FB et al. (2003) Unbiased whole‐genome amplification directly from clinical samples. Genome Research 13: 954–964.

Innis MA, Myambo KB, Gelfand DH and Brow MAD (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction‐amplified. Proceedings of the National Academy of Sciences of the USA 85: 9436–9440.

International Human Genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature 431: 931–945.

Janczewski DN, Yuhki N, Gilbert DA, Jefferson GT and O'Brien SJ (1992) Molecular phylogenetic inference from saber‐toothed cat fossils of Rancho La Brea. Proceedings of the National Academy of Sciences of the USA 89: 9769–9773.

Knight J (2003) Promega changes tack in battle over patent. Nature 426: 373.

Kwoh DY, Davis GR, Whitfield KM et al. (1989) Transcription‐based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead‐based sandwich hybridization format. Proceedings of the National Academy of Sciences of the USA 86: 1173–1177.

Kwok S and Higuchi R (1989) Avoiding false positives with PCR. Nature 339: 237–238.

Lage JM, Leamon JH, Pejovic T et al. (2003) Whole genome analysis of genetic alterations in small DNA samples Using hyperbranched strand displacement amplification and array‐CGH. Genome Research 13: 294–307.

Landegren U, Kaiser R, Sanders J and Hood L (1988) A ligase‐mediated gene detection technique. Science 241: 1077–1080.

Lasken RS and Egholm M (2003) Whole genome amplification: abundant supplies of DNA from precious samples or clinical specimens. Trends in Biotechnology 21: 531–535.

Lee CC, Wu X, Gibbs RA et al. (1988) Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. Science 239: 1288–1291.

Lizardi PM, Huang X, Zhu Z et al. (1998) Mutation detection and single‐molecule counting using isothermal rolling‐circle amplification. Nature Genetics 19: 225–232.

Longo MC, Berninger MS and Hartley JL (1990) Use of uracil DNA glycosylase to control carry‐over contamination in polymerase chain reactions. Gene 93: 125–128.

Margulies M, Egholm M, Altman WE et al. (2005) Genome sequencing in microfabricated high‐density picolitre reactors. Nature 437: 376–380.

Metzker ML, Allain KM and Gibbs RA (1995) Accurate determination of DNA in agarose gels using the novel algorithm GelScann(1.0). Computer Applications in the Biosciences 11: 187–195.

Metzker ML, Mindell DP, Liu XM et al. (2002) Molecular evidence of HIV‐1 transmission in a criminal case. Proceedings of the National Academy of Sciences of the USA 99: 14292–14297.

Mullis KB and Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase‐catalysed chain reaction. Methods in Enzymology 155: 335–351.

Orita M, Iwahana H, Kanazawa H, Hayashi K and Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single‐strand conformation polymorphisms. Proceedings of the National Academy of Sciences of the USA 86: 2766–2770.

Pääbo S (1989) Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. Proceedings of the National Academy of Sciences of the USA 86: 1939–1943.

Pääbo S, Gifford JA and Wilson AC (1988) Mitochondrial DNA sequences from a 7000‐year old brain. Nucleic Acid Research 16: 9775–9787.

Pask R, Rance HE, Barratt BJ et al. (2004) Investigating the utility of combining Φ29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray™ genotyping. BMC Biotechnology 4: 15.

Pinard R, de Winter A, Sarkis GJ et al. (2006) Assessment of whole genome amplification‐induced bias through high‐throughput, massively parallel whole genome sequencing. BMC Genomics 7: 216.

Saiki RK, Gelfand DH, Stoffel S et al. (1988) Primer‐directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491.

Saiki RK, Scharf S, Faloona F et al. (1985) Enzymatic amplification of β‐globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 230: 1350–1354.

Saiki RK, Walsh PS, Levenson CH and Erlich HA (1989) Genetic analysis of amplified DNA with immobilized sequence‐specific oligonucleotide probes. Proceedings of the National Academy of Sciences of the USA 86: 6230–6234.

Sarkar G and Sommer SS (1990) Shedding light on PCR contamination. Nature 343: 27.

Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE and Fanning TG (1997) Initial genetic characterization of the 1918 ‘Spanish’ influenza virus. Science 275: 1793–1796.

Tindall KR and Kunkel TA (1988) Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27: 6008–6013.

Tyagi S, Landegren U, Tazi M, Lizardi PM and Kramer FR (1996) Extremely sensitive, background‐free gene detection using binary probes and Qβ‐replicase. Proceedings of the National Academy of Sciences of the USA 93: 5395–5400.

Wang AM, Doyle MV and Mark DF (1989) Quantitation of mRNA by the polymerase chain reaction. Proceedings of the National Academy of Sciences of the USA 86: 9717–9721.

Wang DG, Fan JB, Siao CJ et al. (1998) Large‐scale identification, mapping, and genotyping of single‐nucleotide polymorphisms in the human genome. Science 280: 1077–1082.

Wheeler DA, Srinivasan M, Egholm M et al. (2008) The complete genome of an individual by massively parallel DNA sequencing. Nature 452: 872–876.

Further Reading

Erlich HA, Gelfand D and Sninsky JJ (1991) Recent advances in the polymerase chain reaction. Science 252: 1643–1650.

Innis MA, Gelfand DH, Sninsky JJ and White TJ (eds) (1990) PCR Protocols: A Guide to Methods and Applications. San Diego: Academic Press.

Mullis KB, Ferré F and Gibbs RA (eds) (1994) The Polymerase Chain Reaction. Boston: Birkhäuser.

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How to Cite close
Metzker, Michael L, and Caskey, C Thomas(Dec 2009) Polymerase Chain Reaction (PCR). In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000998.pub2]