PCR (Polymerase Chain Reaction)

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Overview

Polymerase chain reaction (PCR) is a technique used to exponentially amplify a specific target DNA sequence, allowing for the isolation, sequencing, or cloning of a single sequence among many. PCR was developed in 1983 by Kary Mullis, who received a Nobel Prize in chemistry in 1993 for his invention. The polymerase chain reaction has been elaborated in many ways since its introduction and is now commonly used for a wide variety of applications including genotyping, cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing.

Typically, a PCR is a three-step reaction. The sample containing a dilute concentration of template DNA is mixed with a heat-stable DNA polymerase, such as Taq polymerase, primers, deoxynucleoside triphosphates (dNTPs), and magnesium. In the first step of PCR, the sample is heated to 95–98°C, which denatures the double-stranded DNA, splitting it into two single strands. In the second step, the temperature is decreased to approximately 55–65°C, allowing the primers to bind, or anneal, to specific sequences of DNA at each end of the target sequence, also known as the template. In the third step, the temperature is typically increased to 72°C, allowing the DNA polymerase to extend the primers by the addition of dNTPs to create a new strand of DNA, thus doubling the quantity of DNA in the reaction. This sequence of denaturation, annealing, and extension is repeated for many cycles, resulting in the exponential amplification of the template DNA. As the DNA polymerase loses activity or the dNTPs and primers are consumed, the reaction rate reaches a plateau.

This section includes considerations for the proper design and optimization, analysis, and troubleshooting of polymerase chain reactions, as well as descriptions of the required PCR instrumentation and reagents.

 
 
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Further Reading

Bagasra O et al. (1992). Detection of human immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction. New Engl J Med 326, 1385–1391. PMID: 1569974

de Bruijn MH (1988). Diagnostic DNA amplification: No respite for the elusive parasite. Parasitol Today 4, 293–295. PMID: 15463008

Eckert KA and Kunkel TA (1993). DNA polymerase fidelity and the polymerase chain reaction. PCR Methods Appl 1, 17–24. PMID: 1842916

Hayashi K (1994). PCR-SSCP analysis and its application to DNA diagnosis. Fukuoka Igaku Zaashi 85, 74–77. PMID: 1842918

Higuchi R et al. (1988). A general method of in vitro preparation and specific mutagenesis of DNA fragments: Study of protein and DNA interactions. Nucleic Acids Res 16, 7351–7367. PMID: 3045756

Lee CC et al. (1988). Generation of cDNA probes directed by amino acid sequence: Cloning of urate oxidase. Science 239, 1288–1291. PMID: 3344434

Mullis K et al. (1992). Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction. 1986. Biotechnology 4, 17–27. PMID: 1422010

Ochman H et al. (1988). Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623. PMID: 2852134

Wang Y et al. (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res 32, 1197–1207. PMID: 14973201

Welsh J and McClelland M (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18, 7213–7218. PMID: 2259619

 

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From Plants to Sequences: A Six-Week College Biology Lab Course
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PCR was developed in 1983 by Kary Mullis, who received a Nobel Prize in chemistry in 1993 for his invention. The polymerase chain reaction has been elaborated in many ways since its introduction and is now commonly used for a wide variety of applications including genotyping, cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing.</p> <p>Typically, a PCR is a three-step reaction. The sample containing a dilute concentration of template DNA is mixed with a heat-stable DNA polymerase, such as Taq polymerase, primers, deoxynucleoside triphosphates (dNTPs), and magnesium. In the first step of PCR, the sample is heated to 95&ndash;98&deg;C, which denatures the double-stranded DNA, splitting it into two single strands. In the second step, the temperature is decreased to approximately 55&ndash;65&deg;C, allowing the primers to bind, or anneal, to specific sequences of DNA at each end of the target sequence, also known as the template. In the third step, the temperature is typically increased to 72&deg;C, allowing the DNA polymerase to extend the primers by the addition of dNTPs to create a new strand of DNA, thus doubling the quantity of DNA in the reaction. This sequence of denaturation, annealing, and extension is repeated for many cycles, resulting in the exponential amplification of the template DNA. As the DNA polymerase loses activity or the dNTPs and primers are consumed, the reaction rate reaches a plateau.</p> <p>This section includes considerations for the proper <a href="/evportal/destination/solutions?catID=LUSO1VKG4">design and optimization</a>, <a href="/evportal/destination/solutions?catID=LUSO2PESH">analysis</a>, and <a href="/evportal/destination/solutions?catID=LUSO3HC4S">troubleshooting</a> of polymerase chain reactions, as well as descriptions of the required PCR <a href="/evportal/destination/solutions?catID=LUSNZ1E8Z">instrumentation</a> and <a href="/evportal/destination/solutions?catID=LUSO0V4VY">reagents</a>.</p> Further Reading <p>Bagasra O et al. (1992). Detection of human immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction. New Engl J Med 326, 1385&ndash;1391. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/1569974" target="_blank" rel="noopener noreferrer">1569974</a></p> <p>de Bruijn MH (1988). Diagnostic DNA amplification: No respite for the elusive parasite. Parasitol Today 4, 293&ndash;295. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/15463008" target="_blank" rel="noopener noreferrer">15463008</a></p> <p>Eckert KA and Kunkel TA (1993). DNA polymerase fidelity and the polymerase chain reaction. PCR Methods Appl 1, 17&ndash;24. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/1842916" target="_blank" rel="noopener noreferrer">1842916</a></p> <p>Hayashi K (1994). PCR-SSCP analysis and its application to DNA diagnosis. Fukuoka Igaku Zaashi 85, 74&ndash;77. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/1842918" target="_blank" rel="noopener noreferrer">1842918</a></p> <p>Higuchi R et al. (1988). A general method of in vitro preparation and specific mutagenesis of DNA fragments: Study of protein and DNA interactions. Nucleic Acids Res 16, 7351&ndash;7367. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/3045756" target="_blank" rel="noopener noreferrer">3045756</a></p> <p>Lee CC et al. (1988). Generation of cDNA probes directed by amino acid sequence: Cloning of urate oxidase. Science 239, 1288&ndash;1291. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/3344434" target="_blank" rel="noopener noreferrer">3344434</a></p> <p>Mullis K et al. (1992). Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction. 1986. Biotechnology 4, 17&ndash;27. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/1422010" target="_blank" rel="noopener noreferrer">1422010</a></p> <p>Ochman H et al. (1988). Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621&ndash;623. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/2852134" target="_blank" rel="noopener noreferrer">2852134</a></p> <p>Wang Y et al. (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res 32, 1197&ndash;1207. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/14973201" target="_blank" rel="noopener noreferrer">14973201</a></p> <p>Welsh J and McClelland M (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18, 7213&ndash;7218. 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