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Optimizing pfuturbo DNA Polymerase Amplification Reactions with,,,Perfect Match PCR Enhancer

ct Match enhancer, PfuTurbo DNA polymerase synthesized multiple products, including the desired 6.2-kb band, while Taq DNA polymerase generated a smear (Figure 1). However, adding Perfect Match enhancer dramatically increased the yield of desired product in both the PfuTurbo polymerase and Taq amplification reactions (Figure 1). In the PfuTurbo polymerase plus Perfect Match enhancer reaction, undesired side products were still produced, but the relative proportions of each product were altered such that the percentage of desired product (6.2 kb) dramatically increased. Perfect Match enhancer was required for obtaining the Taq generated product, and it also greatly reduced smearing. The lower product yield achieved with Taq DNA polymerase is consistent with Taqs reduced target-length capability complared to PfuTurbo DNA polymerase.

Successful Amplification of Long Targets

The second amplification system tested (Figure 2) was designed to amplify a 6.6-kb fragment from the same template described in Figure 1. In the absence of Perfect Match enhancer, PfuTurbo DNA polymerase synthesized a small amount of a shorter undesired product, while Taq DNA polymerase produced a smear. In the presence of Perfect Match enhancer, both enzymes produced the specific product but product yield was higher for the PfuTurbo amplification.In cases where Perfect Match enhancer must be present to amplify relatively long targets (e.g., Figure 1 and Figure 2), it may act by reducing secondary structure formation in the template strand, which impedes translocation of the DNA polymerase.5

Improved Specificity

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