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Comparing Fidelity and Performance of Proofreading PCR Enzymes

Turbo and Pfx DNA polymerases using shortened PCR extension times. In Figure 5, PCR reactions were carried out as described above, except that extension times of 15 seconds/kb were used. Under these conditions, PfuTurbo DNA polymerase was found to synthesize significantly higher yield of a 1.9-kb product than Pfx DNA polymerase. Thus, despite the higher processivity and polymerization rate reported for KOD DNA polymerase,6 Pfu DNA polymerase can produce higher product yields, in a shorter time, when combined with the Turbo PCR enhancing factor.

Room-Temperature PCR Reaction Assembly

All PCR amplifications with PfuTurbo DNA polymerase, including those described here and in other Strategies articles,4,5 are assembled at room temperature. Previously, we showed that Pfu DNA polymerase exhibits only 0.7% and 3.9% maximal polymerase activity at 25C and 45C, respectively.11 In contrast, Taq DNA polymerase exhibits 2.6% activity at 25C (room temperature) and 44% activity at 45C.11 Reduced activity at low temperatures encountered during PCR is thought to contribute to higher amplification specificity achieved with Pfu DNA polymerase, as compared to Taq DNA polymerase.11 Various hot start procedures have been developed for Taq-based PCR to reduce mispaired primer extension that results in the synthesis of nonspecific background. The benefits of hot start modifications (e.g., neutralizing antibodies) have not been directly demonstrated for PCR enzymes derived from hyperthermophilic archaea (e.g. Pfu DNA polymerase, KOD, Deep Vent). Although Pfx DNA polymerase is blended with neutralizing antibody, the results shown here for 10 d
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