PCR FIDELITY OF PFU DNA-POLYMERASE AND OTHER THERMOSTABLE DNA-POLYMERASES

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UITma DNA polymerases were compared using a PCR-based forward mutation assay, Av erage error rates (mutation frequency/bp/duplication) increased as fol lows: 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 UITma (similar to 5 x 10(-5 )). Buffer optimization experiments indicated that Pfu fidelity was hi ghest in the presence of 2-3 mM MgSO4 and 100-300 mu M each dNTP and a t pH 8.5-9.1. Under these conditions, the error rate of exo(-) Pfu was similar to 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 decr eased similar to 2-fold, while the error rate of exo(-) Pfu increased similar to 9-fold. An increase in error rate with pH has also been not ed for the exonuclease-deficient DNA polymerases Taq and exo(-) Klenow , suggesting that the parameters which influence replication error rat es may be similar in pol I- and alpha-like polymerases. Finally, the f idelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polyme rase mixture were found to be less than the error rate of Taq DNA poly merase, but similar to 3-4-fold higher than the error rate of Pfu DNA polymerase.