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Pfu DNA Polymerase as a Hot Start Alternative

bitor9 until a stringent primer-annealing temperature has been attained (usually 60 to 95C).

PCR Comparisons with Hot Start Techniques

Since hot start improvements are most noticeable when amplifying complex, low copy number targets,6-8 we performed PCR comparisons using several genomic DNA targets that tend to generate troublesome cold start artifacts. The performance of Pfu DNA polymerase was compared to Taq DNA polymerase alone, Taq DNA polymerase in conjunction with various hot start techniques or AmpliTaq Gold DNA polymerase, which is a heat-activated version of AmpliTaq enzyme. The results from amplifying a 2-kb target from a single copy region of a transgenic mouse genome are shown (figure 2A). PCR amplifications used equivalent polymerase units of Taq DNA polymerase (alone or in conjunction with a Taq DNA polymerase-specific antibody or magnesium wax beads), AmpliTaq Gold DNA polymerase or Pfu DNA polymerase. Similar comparisons were performed using a 120-bp target in the human fucosidase gene (figure 2B) and a 230-bp target from the Epstein-Barr virus genome (figure 2C).

The PCR results shown in figure 2A, 2B and 2C demonstrate that specificity and yield of the desired amplification target are improved to varying extents when hot start techniques are used with Taq DNA polymerase (in figure 2A, 2B and 2C, compare lanes 3-8 to lanes 1, 2). For the 2-kb target (figure 2A), undesired PCR products were eliminated when Taq DNA polymerase amplifications were performed in conjunction with Taq DNA polymerase-specific antibody or magnesium wax beads. The 2-kb target, however, could not be amp
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