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Comparing Fidelity and Performance of Proofreading PCR Enzymes

was 2.7-fold higher than the mean error rate of PfuTurbo DNA polymerase (Figure 1). Moreover, we observed no significant differences in error rate when Pfx DNA polymerase was used in cloned Pfu PCR buffer (data not show), indicating that relatively poor fidelity is an intrinsic property of KOD DNA polymerase.

PCR Yield and Target-Length Comparisons

The PCR performance of Pfx DNA polymerase was then evaluated with respect to product yield and target-length capability. PfuTurbo DNA polymerase and Pfx DNA polymerase were used to amplify a panel of complex, genomic DNA targets, ranging in length from 0.9 kb to 19 kb. Standard reaction conditions were employed, including the use of 100 to 200 ng of template, 30 PCR cycles, 1 minute per kb extension times, and room-temperature reaction assemblies (see below). Amplifications were performed under identical conditions (Methods), with the following exceptions: As per manufacturers recommendations, Pfx PCRs were carried out with 300 M each dNTP, 1.25 U of enzyme and 68C extension temperatures, while PfuTurbo DNA polymerase amplifications employed 200 M (<10 kb) or 500 M (>10 kb) each dNTP, 2.5 U (<12 kb) or 5 U (>12 kb) of enzyme, and 72C extension temperatures.

Fig.2

In all comparisons, PfuTurbo DNA polymerase produced higher product yields than Pfx DNA polymerase (Figure 2). Moreover, synthesis of the four longest 9.3-kb to 19-kb targets was achieved with PfuTurbo DNA polymerase, but not with Pfx DNA polymerase. As described , amplification of long genomic targets (>9 kb) by PfuTurbo DNA polymerase is limited by buffer components, rather than the robustness of the enzyme. Increasing dNTPs (to 500 M) and PCR buffer concentration (to 1.5X) was sufficient to allow PfuTurbo DNA pol
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