( MSO) that can be potentially damaging t...)Pfu DNA Polymerase as a Hot Start Alternative

MSO) that can be potentially damaging to the DNA template or to polymerase fidelity^10,11 and (4) minimal polymerase activity at temperatures at or below 50^oC, which may contribute to fewer undesirable PCR artifacts. Pfu DNA polymerase provides an alternative to Taq DNA polymerase, alone or in conjunction with a hot start technique, for increasing the yield and specificity of PCR products.

Methods

The DNA polymerase activity of Taq and Pfu DNA polymerases was measured by monitoring the incorporation of [³H]TTP into high-molecular-weight DNA using primed M13mp18(+) single-stranded template. The 40-base primer (54% GC) anneals to the multiple cloning region. Primer-extension reactions were carried out in each enzymes recommended PCR buffer in the presence of 200 M dATP, dCTP and dGTP, 200 M TTP (including 10 M [³H]TTP; specific activity of 20.9 Ci/mmole) and 1 g primer-annealed M13 DNA per 100-l reaction. Reactions were preincubated at the indicated temperatures, and polymerization was initiated with the addition of cloned Taq or cloned Pfu DNA polymerase to a final concentration of 10 U per 200-l reaction. (Unit concentrations of Taq and Pfu DNA polymerases were determined in the same assay by measuring nucleotide incorporation into activated calf thymus DNA at 72C.) After 10 minutes, polymerization was stopped by transferring the reactions to ice. From each reaction, 5 l was transferred to a chilled DE81 DNA-binding filter. Unincorporated [³H]TTP was removed by washing the filters in 8 changes of 2X SSC. Filters were added to scintillation cocktail and incorporated radioactivity was measured. Previous assays had demonstrated that the polymerase concentrations and extension time used for these reactions gave incorporation values that fell within the linear range of the assay for each reaction temperature. The mean % activity ( + one stand
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