( lified with AmpliTaq Gold DNA polymeras...)Pfu DNA Polymerase as a Hot Start Alternative

lified with AmpliTaq Gold DNA polymerase under the conditions used. For the 120-bp target (figure 2B), extraneous products were synthesized with Taq DNA polymerase but not with AmpliTaq Gold DNA polymerase. The addition of Taq DNA polymerase-specific antibody or the magnesium wax beads did not eliminate the synthesis of artifacts. Finally, with the 230-bp target (figure 2C), undesired PCR products were absent when amplifications were conducted with Taq2000 DNA polymerase in the presence of magnesium wax beads or with AmpliTaq Gold DNA polymerase; however, PCR product yield was significantly reduced in reactions using AmpliTaq Gold DNA polymerase (lanes 5-6). For the 230-bp target, the addition of Taq DNA polymerase-specific antibody did not reduce the synthesis of undesired products by Taq DNA polymerase.

In total, our results demonstrate the variability of hot start techniques in eliminating the synthesis of undesired products by Taq DNA polymerase. In contrast, Pfu DNA polymerase (in figure 2A, 2B and 2C, lanes 9 and 10) consistently generated only the desired PCR product, and yields of PCR products were comparable to those generated with the best Taq DNA polymerase plus hot start combinations (figure 2A, lanes 3, 4, 7 and 8; figure 2B, lanes 5, 6; figure 2C, lanes 7, 8). The relatively poor performance of hot start techniques for amplifying the 120-bp and 230-bp targets suggests that mispaired primer-extension reactions may be occurring at primer-annealing steps that take place after the first PCR cycle. Of course, current hot start techniques are ineffective after the initial denaturation step. The use of Pfu DNA polymerase,
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