Polymerase Chain Reaction (PCR): Design and Optimization of Reactions

Abstract

Optimization of the polymerase chain reaction (PCR) includes the consideration of buffer, additives, magnesium chloride, deoxynucleotides, primer design, thermostable DNA polymerase, DNA template, reaction volume, denaturation time, annealing temperature and extension time. Nonspecific products can be further minimized by using a ‘hot start’ and a variable annealing temperature (touchdown PCR).

Keywords: PCR; primer design; hot start; annealing temperature; touchdown PCR

References

Chou Q, Russell M, Birch DE, Raymond J and Bloch W (1992) Prevention of pre‐PCR mis‐priming and primer dimerization improves low‐copy‐number amplifications. Nucleic Acids Research 20: 1717–1723.

Kolmodin LA and Williams JF (1997) PCR: basic principles and routine practice. In: White BA (ed.) PCR Cloning Protocols. From Molecular Cloning to Genetic Engineering, pp. 3–16. Totowa, NJ: Humana Press.

Preston GM (1997) Cloning gene family members using PCR with degenerate oligonucleotide primers. In: White BA (ed.) PCR Cloning Protocols. From Molecular Cloning to Genetic Engineering, pp. 433–450. Totowa, NJ: Humana Press.

Roux KA and Hecker KH (1997) One‐step optimization using touchdown and stepdown PCR. In: White BA (ed.) PCR Cloning Protocols. From Molecular Cloning to Genetic Engineering, pp. 39–46. Totowa, NJ: Humana Press.

Rychlik W (1993) Selection of primers for polymerase chain reaction. In: White BA (ed.) PCR Protocols. Current Methods and Applications, pp. 31–40. Totowa, NJ: Humana Press.

Sarkar G, Kapelner S and Sommer SS (1990) Formamide can dramatically improve the specificity of PCR. Nucleic Acids Research 18: 7465.

Further Reading

Bartlett JMS and Stirling D (eds.) (2003) PCR Protocols, 2nd edn. Totowa, NJ: Humana Press.

White BA (ed.) (1996) PCR Cloning Protocols. Totowa, NJ: Humana Press.

Newton CR (1995) PCR: Essential Data Series. New York: John Wiley & Sons.

Dieffenbach C and Dveksler GS (1995) PCR Primer: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Press.

Griffin, HG and Griffin AM (1994) PCR Technology, Current Innovations. Boca Raton, FL: CRC Press.

Web Links

Primer 3 http://www‐genome.wi.mit.edu/cgi‐bin/primer/primer3_www.cgi

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How to Cite close
White, Bruce A(Sep 2005) Polymerase Chain Reaction (PCR): Design and Optimization of Reactions. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0005340]