PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases

TLDR

Error rates of exonuclease‑deficient polymerases such as Taq and exo‑Klenow rise with pH, indicating that factors affecting replication fidelity are likely shared across pol I‑ and α‑like polymerases. The authors compared the replication fidelities of Pfu, Taq, Vent, Deep Vent, and UlTma polymerases using a PCR‑based forward mutation assay and also evaluated the fidelity of long‑PCR DNA polymerase mixtures. Pfu exhibited the lowest error rate (1.3 × 10⁻⁶ bp⁻¹), followed by Deep Vent (2.7 × 10⁻⁶), Vent (2.8 × 10⁻⁶), Taq (8.0 × 10⁻⁶), and exo‑Pfu/UlTma (~5 × 10⁻⁵); buffer optimization (2–3 mM MgSO₄, 100–300 µM dNTPs, pH 8.5–9.1) maximized Pfu fidelity, while increasing pH reduced Pfu error but increased exo‑Pfu error, and long‑PCR mixtures had error rates between Taq and Pfu.

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 × 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.