Optimized PCR using Vent polymerase.

PCR has become an essential tool of the molecular biologist, and substantial literature exists characterizing the performance of Taq polymerase in this reaction. (~-4) Use of a thermostable polymerase that, unlike Taq, possesses 3'---> 5' exonuclease activity is desirable in situations where higher fidelity and/ or blunt-end products are required. (s--8) Vent polymerase is one such enzyme, but information on optimal use of this enzyme in PCR is limited. Ling et al. identified conditions that optimize the fidelity of PCR using Vent polymerase. (s) We commonly need high-fidelity blunt PCR products for use in DNA constructions. Our initial experience with Vent polymerase convinced us that, within the previously defined conditions that optimize fidelity, careful optimization for PCR product quantity in addition to quality was essential. Therefore, we systematically examined a number of potentially important PCR conditions in a stepwise manner. This analysis led to a highly optimized procedure described below.