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

Bruce A White, University of Connecticut Health Center, Farmington, Connecticut, USA

Published online: September 2005

DOI: 10.1038/npg.els.0005340

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.
    book 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.
    book 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.
    book 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.
    book 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
    book Bartlett JMS and Stirling D (eds.) (2003) PCR Protocols, 2nd edn. Totowa, NJ: Humana Press.
    book White BA (ed.) (1996) PCR Cloning Protocols. Totowa, NJ: Humana Press.
    book Newton CR (1995) PCR: Essential Data Series. New York: John Wiley & Sons.
    book Dieffenbach C and Dveksler GS (1995) PCR Primer: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Press.
    book Griffin, HG and Griffin AM (1994) PCR Technology, Current Innovations. Boca Raton, FL: CRC Press.
 Web Links
    ePath Primer 3 http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi

Related Sub-Topics:

Replication and Recombination | Techniques and Tools in Clinical Genetics | Techniques and Tools in Molecular Biology
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Article Title: Polymerase Chain Reaction (PCR): Design and Optimization of Reactions
 
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]