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Nucleic Acids Res. 1990 November 11; 18(21): 6409–6412.
PMCID: PMC332522

Optimization of the annealing temperature for DNA amplification in vitro.


In the polymerase chain reaction (PCR) technique, DNA is amplified in vitro by a series of polymerization cycles consisting of three temperature-dependent steps: DNA denaturation, primer-template annealing, and DNA synthesis by a thermostable DNA polymerase. The purity and yield of the reaction products depend on several parameters, one of which is the annealing temperature (Ta). At both sub- and super-optimal Ta values, non-specific products may be formed, and the yield of products is reduced. Optimizing the Ta is especially critical when long products are synthesized or when total genomic DNA is the substrate for PCR. In this article we experimentally determine the optimal annealing temperature (TaOPT) values for several primer-template pairs and develop a method for its calculation. The TaOPT is found to be a function of the melting temperatures of the less stable primer-template pair and of the product. The fact that experimental and calculated TaOPT values agree to within 0.7 degree C eliminates the need for determining TaOPT experimentally. Synthesis of DNA fragments shorter than 1 kb is more efficient if a variable Ta is used, such that the Ta is higher in each consecutive cycle.

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Selected References

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  • Guyer RL, Koshland DE., Jr The Molecule of the Year. Science. 1989 Dec 22;246(4937):1543–1546. [PubMed]
  • Rychlik W, Rhoads RE. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res. 1989 Nov 11;17(21):8543–8551. [PMC free article] [PubMed]
  • Hiremath LS, Hiremath ST, Rychlik W, Joshi S, Domier LL, Rhoads RE. In vitro synthesis, phosphorylation, and localization on 48 S initiation complexes of human protein synthesis initiation factor 4E. J Biol Chem. 1989 Jan 15;264(2):1132–1138. [PubMed]
  • Borer PN, Dengler B, Tinoco I, Jr, Uhlenbeck OC. Stability of ribonucleic acid double-stranded helices. J Mol Biol. 1974 Jul 15;86(4):843–853. [PubMed]
  • Breslauer KJ, Frank R, Blöcker H, Marky LA. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3746–3750. [PubMed]
  • Schildkraut C. Dependence of the melting temperature of DNA on salt concentration. Biopolymers. 1965;3(2):195–208. [PubMed]
  • Freier SM, Kierzek R, Jaeger JA, Sugimoto N, Caruthers MH, Neilson T, Turner DH. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. [PubMed]
  • Baldino F, Jr, Chesselet MF, Lewis ME. High-resolution in situ hybridization histochemistry. Methods Enzymol. 1989;168:761–777. [PubMed]
  • Innis MA, Myambo KB, Gelfand DH, Brow MA. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9436–9440. [PubMed]

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