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Comparing Fidelity and Performance of Proofreading PCR Enzymes

ymerase to synthesize complex genomic targets up to 19 kb in length. In comparison, Pfx DNA polymerase could not successfully amplify the 17-kb and 19-kb targets in the presence of 500 M each dNTP and 0.5X to 2X concentrations of Pfx PCR buffer .

Sensitivity Comparisons

Fig.3

Next, we compared PfuTurbo DNA polymerase and Pfx DNA polymerase with respect to sensitivity. Using the standard PCR reaction conditions described above, PfuTurbo DNA polymerase synthesized a 4-kb product from as little as 10 fg of lambda DNA (Figure 3). In contrast, at least 5 ng of lambda DNA was consistently required for amplifications carried out using Pfx DNA polymerase (Figure 3, data not shown). Despite the inclusion of polymerase-neutralizing antibodies, Pfx DNA polymerase produced additional background bands in this amplification system, which were absent or greatly reduced in PCRs carried out with PfuTurbo DNA polymerase (Figure 3).

Fig.4

The superior sensitivity of PfuTurbo DNA polymerase is further demonstrated in Figure 4, where PfuTurbo DNA polymerase synthesized high yields of a 4-kb product from as little as 25 ng of genomic DNA. In contrast, Pfx DNA polymerase required at least 50 ng of genomic DNA, and product yields were significantly lower than those produced by PfuTurbo DNA polymerase (Figure 4). In studies employing a 6-kb genomic target, PfuTurbo DNA polymerase again exhibited superior sensitivity, compared to Pfx DNA polymerase (Figure 4).

Rate Comparisons

Fig.5

Finally, we compared product yields produced with Pfu
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