• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. Sep 15, 1996; 24(18): 3546–3551.
PMCID: PMC146123

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

Abstract

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.

Full Text

The Full Text of this article is available as a PDF (113K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Lundberg KS, Shoemaker DD, Adams MW, Short JM, Sorge JA, Mathur EJ. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene. 1991 Dec 1;108(1):1–6. [PubMed]
  • Kong H, Kucera RB, Jack WE. Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities. J Biol Chem. 1993 Jan 25;268(3):1965–1975. [PubMed]
  • Flaman JM, Frebourg T, Moreau V, Charbonnier F, Martin C, Ishioka C, Friend SH, Iggo R. A rapid PCR fidelity assay. Nucleic Acids Res. 1994 Aug 11;22(15):3259–3260. [PMC free article] [PubMed]
  • Barnes WM. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216–2220. [PMC free article] [PubMed]
  • Echols H, Goodman MF. Fidelity mechanisms in DNA replication. Annu Rev Biochem. 1991;60:477–511. [PubMed]
  • Kunkel TA. DNA replication fidelity. J Biol Chem. 1992 Sep 15;267(26):18251–18254. [PubMed]
  • Goodman MF, Creighton S, Bloom LB, Petruska J. Biochemical basis of DNA replication fidelity. Crit Rev Biochem Mol Biol. 1993;28(2):83–126. [PubMed]
  • Kunkel TA. Exonucleolytic proofreading. Cell. 1988 Jun 17;53(6):837–840. [PubMed]
  • Mattila P, Korpela J, Tenkanen T, Pitkänen K. Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 1991 Sep 25;19(18):4967–4973. [PMC free article] [PubMed]
  • Ling LL, Keohavong P, Dias C, Thilly WG. Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and vent DNA polymerases. PCR Methods Appl. 1991 Aug;1(1):63–69. [PubMed]
  • Eckert KA, Kunkel TA. High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res. 1990 Jul 11;18(13):3739–3744. [PMC free article] [PubMed]
  • Eckert KA, Kunkel TA. Effect of reaction pH on the fidelity and processivity of exonuclease-deficient Klenow polymerase. J Biol Chem. 1993 Jun 25;268(18):13462–13471. [PubMed]
  • Provost GS, Kretz PL, Hamner RT, Matthews CD, Rogers BJ, Lundberg KS, Dycaico MJ, Short JM. Transgenic systems for in vivo mutation analysis. Mutat Res. 1993 Jul;288(1):133–149. [PubMed]
  • Good NE, Winget GD, Winter W, Connolly TN, Izawa S, Singh RM. Hydrogen ion buffers for biological research. Biochemistry. 1966 Feb;5(2):467–477. [PubMed]
  • Clayton LK, Goodman MF, Branscomb EW, Galas DJ. Error induction and correction by mutant and wild type T4 DNA polymerases. Kinetic error discrimination mechanisms. J Biol Chem. 1979 Mar 25;254(6):1902–1912. [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. [PMC free article] [PubMed]
  • Huang MM, Arnheim N, Goodman MF. Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. Nucleic Acids Res. 1992 Sep 11;20(17):4567–4573. [PMC free article] [PubMed]
  • Mathur EJ, Adams MW, Callen WN, Cline JM. The DNA polymerase gene from the hyperthermophilic marine archaebacterium, Pyrococcus furiosus, shows sequence homology with alpha-like DNA polymerases. Nucleic Acids Res. 1991 Dec 25;19(24):6952–6952. [PMC free article] [PubMed]
  • Hu G. DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment. DNA Cell Biol. 1993 Oct;12(8):763–770. [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...