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Comparing Fidelity and Performance of Proofreading PCR Enzymes

old lower than those of DNA polymerase mixtures,1,9 and 6- to 12-fold lower than that of Taq DNA polymerase.1,10 Using the same lacI PCR mutation assay, PfuTurbo DNA polymerase exhibited the same high fidelity as cloned Pfu DNA polymerase.4

DNA polymerase fidelity is expressed as error ratethe mutation frequency per base pair per duplication (MF/bp/d). When determining polymerase error rates in PCR-based assays, mutation frequencies [# mutants/total # clones] must be normalized with respect to the number of template doublings (d), as errors accumulate with each template duplication. The number of template doublings [d; determined as 2d = (amount of product)/(amount of template)] is influenced by the amount of starting template, the presence of excess reactants, PCR efficiency, the presence of inhibitors, and the number of cycles performed. Therefore, to compare PCR enzyme fidelity accurately, one must compare intrinsic error rates rather than mutation frequencies, which vary from experiment to experiment.

The mutation frequency of KOD DNA polymerase was determined previously by Tagaki, et al.6, using both gap-filling (single primer extension) and PCR-based forward mutation assays (lacZ target gene). In the gap-filling M13 assay, the mutation frequency of KOD DNA polymerase was, as Tagaki, et al. described, similar to that of Pfu DNA polymerase.6 Unfortunately, PCR error rates could not be calculated from these studies as only mutation frequencies were reported.

Fig.1

To assess the fidelity of Pfx DNA polymerase, we measured the error rates of Pfx, PfuTurbo, and Taq DNA polymerases in the same assay (Figure 1). PCR fidelity measurements were performed in each manufacturers recommended PCR buffer. The mean error rate of Pfx DNA polymerase
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