( Figure 2A...)Pfu DNA Polymerase as a Hot Start Alternative

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Pfu DNA polymerases low activity at or below 50C may represent one of its most important advantages in PCR. The use of Pfu DNA polymerase should lead to fewer mispaired primer-extension reactions than occur with Taq DNA polymerase. Mismatched primer-extension reactions are most likely to occur during (1) preparation of the PCR reaction mixtures, (2) ramping to the initial denaturation temperature and (3) successive primer-annealing steps (typically performed at 45 to 55C). Undesirable PCR products, which are typically shorter than the desired target, may be preferentially amplified when they become established, subsequently interfering with amplification, detection and quantification of the desired target. Since Taq DNA polymerase exhibits significantly higher polymerase activity than Pfu DNA polymerase at lower temperatures (figure 2), Taq DNA polymerase has a greater potential for extending mispaired primers that anneal during these three low-temperature phases of PCR amplification.

Hot start PCR protocols have been developed to reduce or prevent mispaired primer-extension reactions from occurring prior to the initial denaturation step in Taq DNA polymerase-based PCR amplifications.^6,7 In these protocols, a critical PCR component (polymerase, magnesium or dNTPs) is manually withheld by separation from the reaction by a wax barrier,⁸ or the polymerase is reversibly inactivated by binding to an antibody, oligonucleotide or protein inhi
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