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Comparing Fidelity and Performance of Proofreading PCR Enzymes

ifferent amplification systems, of varying lengths and complexities, indicate that PfuTurbo DNA polymerase provides comparable, if not superior (Figure 3), specificity compared to Platinum Pfx DNA polymerase.

Conclusions

Stratagenes PfuTurbo DNA polymerase is the superior choice for all high-fidelity PCR applications requiring the highest performance possible. It exhibits unparalleled PCR fidelity and performance compared to a competitors Platinum Pfx DNA polymerase. The intrinsic error rate of PfuTurbo DNA polymerase is significantly lower than the error rate of Pfx DNA polymerase. Side-by-side comparisons show that PfuTurbo DNA polymerase produces higher product yields, while exhibiting greater sensitivity and target-length capability, compared to Pfx DNA polymerase.

Methods

The PCR fidelity assay was carried out as described,1 using cloned PfuTurbo DNA polymerase (Stratagene), Platinum Pfx DNA polymerase (LTI), and Taq2000 DNA polymerase (Stratagene). For fidelity assays, PCR amplifications were carried out using identical conditions, except that PCRs employed each enzymes recommended PCR buffer.

For PCR comparisons, amplifications employed 100 ng (<12 kb) or 200 ng (>12 kb) of genomic DNA template and 100 ng (<12 kb) or 200 ng (>12 kb) of each primer. 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 PfuTurbo DNA polymerase, and 1X (<9 kb) or 1.5X (>9 kb) PfuTurbo PCR buffer, as recommended. Pfx PCRs employed 300 M each dNTP, 1.25 U of Pfx DNA polymerase, and 1X Pfx PCR buffer, as recommended by the manufacturer. PCR reactions were conducted in a RoboCycler Gradient 96 temperature cycler (Stratagene), fitted with a Hot Top assembly, using 200-l thin-walled PCR tubes. Except as noted, the temperature cycling parameters were as
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