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High-Fidelity PCR with a Novel Polymerase Mixture

relatively high error rate of Taq DNA polymerase has been attributed to the absence of an associated proofreading activity. Taq DNA polymerase exhibits a comparatively rapid rate of nucleotide incorporation, averaging approximately 2800 nucleotides per minute,2 which contributes to the reliability of this enzyme.

Stratagene provides both native Taq DNA polymerase, purified from T. Aquaticus YT1, as well as a fulllength recombinant version, Taq2000 DNA polymerase. Stratagene's highly purified Taq2000 DNA polymerase and associated PCR buffer have been shown to exhibit minimal artifactual smearing, which is commonly seen with competitors Taq DNA polymerase preparations using long (8 minute) extension times.

Pfu DNA Polymerase for Highest Fidelity and Thermostability

Pfu DNA polymerase is purified from the thermophilic archae Pyrococcus furiosus and exhibits both 5- to 3- polymerase and 3- to 5-exonucleasedependent proofreading activities. The proofreading activity associated with Pfu DNA polymerase contributes to its low PCR error rate. Several different methods, including forward mutation assays,4,5,6 DNA sequence analyses7 and denaturant gradient gel electrophoresis (DGGE) analyses8 have been used to show that Pfu DNA polymerase has the lowest error rate of any thermostable DNA polymerase studied to date. Pfu DNA polymerase also lacks terminal transferase activity and produces only blunt-ended PCR products, thereby making Pfu DNA polymerase the enzyme of choice for blunt-end PCR cloning with vectors such as the PCR-Script cloning vectors.9 In addition, Pfu DNA polymerase has unusually high thermostability, retaining more than 95% activity after 1 hour at 95C10 (half-life of approximately 12 hours at 95C) and c
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