Traditional hot start additives unnecessary
Kirk B. Nielson Janice Cline Holly Hogrefe
Stratagene Cloning Systems, Inc.
This article describes the important, novel application of Stratagenes Pfu DNA polymerase for minimizing PCR cold start mismatched primer-extension reactions. These mismatched extension reactions result in the synthesis of undesirable artifactual PCR products. The problem of mismatched extension reactions may be alleviated by using Taq DNA polymerase in conjunction with hot start techniques, which prevent nucleotide incorporation prior to the first PCR denaturation step. Pfu DNA polymerase,, * which exhibits minimal activity at problematic lower temperatures, obviates the need for Taq DNA polymerase-based hot start procedures.
Pfu DNA polymerase has the lowest error rate of any thermostable DNA polymerase studied to date.1,2 This feature makes it the preferred polymerase for techniques that require high-fidelity DNA synthesis by the polymerase chain reaction (PCR). Applications for Pfu DNA polymerase include gene cloning and expression, sequencing and site-directed mutagenesis. Pfu DNA polymerase exhibits exceptionally high thermostability3 and requires a relatively high temperature for optimal activity (Topt75C), characteristics that have significant implications in PCR applications. Most notable is Pfu DNA polymerases relatively low activity, in comparison to other commonly used PCR enzymes, at temperatures ranging from 22 to 50C. Minimal polymerase activity at these temperatures should result in fewer mispaired primer-extension reactions than occur with Taq DNA polymerase, which exhibits significantly higher activity at temperatures used in the primer-annealing phase of PCR (~50C).
The relatively high thermostability and Topt <
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