PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases

Nucleic Acids Res. 1996 Sep 15;24(18):3546-51. doi: 10.1093/nar/24.18.3546.


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.

Publication types

  • Comparative Study

MeSH terms

  • DNA-Directed DNA Polymerase / metabolism*
  • Enzyme Stability
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Polymerase Chain Reaction* / methods
  • Reproducibility of Results
  • Taq Polymerase


  • Deep Vent DNA polymerase
  • Pfu DNA polymerase
  • Taq Polymerase
  • Tli polymerase
  • UlTma DNA polymerase
  • DNA-Directed DNA Polymerase