PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases

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 MgSO 4 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 Klen taq/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.