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Novel PCR Enhancing Factor Improves Performance of Pfu DNA Polymerase

fers.3 In general, however, PCR product yields decrease as the length of an amplicon increases, irrespective of the PCR enzyme used.

The recent development of DNA polymerase mixtures has largely overcome the problem of reduced yields when amplifying longer targets (e.g., >4-kb genomic targets) with single DNA polymerases. For example, Stratagene has developed two DNA polymerase mixtures containing Taq and Pfu DNA polymerases: TaqPlus Long PCR system**,, for amplifying targets up to 35 kb in length4 and TaqPlus Precision PCR system**, for high-fidelity amplification of genomic targets up to 10 kb and cloned targets up to 15 kb.5 DNA polymerase mixtures typically consist of a blend of a nonproofreading DNA polymerase (e.g., Taq DNA polymerase) and a proofreading DNA polymerase (e.g., Pfu DNA polymerase). The addition of the proofreading enzyme is thought to enhance amplification of longer targets by repairing mispaired primer termini that Taq DNA polymerase cannot extend.6 However, because DNA polymerase mixtures are composed of a significant proportion of a nonproofreading DNA polymerase, error rates for mixtures are closer to that of Taq DNA polymerase and approximately 3- to 6-fold higher than the error rate of Pfu DNA polymerase.1,4,5

In a continuing effort to improve high-fidelity PCR performance, Stratagene has discovered a novel thermo- stable factor that enhances the performance of Pfu DNA polymerase in PCR amplifications without altering the enzymes high replication fidelity. This PCR enhancing factor was isolated from Pyrococcus and shown to lack both DNA polymerase and exonuclease activities (data not shown). PfuTurbo DNA polymerase is an optimized blend of cloned Pfu DNA polymerase and this PCR enhancing factor. PfuTurbo
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