PCR Fidelity of Pfu DNA Polymerase and Other Thermostable DNA Polymerases

摘要

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 × 10?6) Deep Vent (2.7 × 10?6) Vent (2.8 × 10?6) Taq (8.0 × 10?6) ? exo? Pfu and UlTma (～5 × 10?5). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2–3 mM MgSO₄ and 100–300 μM each dNTP and at pH 8.5–9.1. Under these conditions, the error rate of exo? Pfu was ～40-fold higher (5 × 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 ～2-fold, while the error rate of exo? Pfu increased ～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 I- and α-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 ～3–4-fold higher than the error rate of Pfu DNA polymerase.