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DNA fragments were amplified from lambda DNA in 50 µL of PCR mixture containing 1.25 to 5 u of Pfu DNA Polymerase (native).
M – ZipRuler Express DNA Ladder Mix
1-9 – PCR products of various lengths
Thermo Scientific Pfu DNA Polymerase is a highly thermostable DNA polymerase from the hyperthermophilic archaeum Pyrococcus furiosus. The enzyme catalyzes the template-dependent polymerization of nucleotides into duplex DNA in the 5’→3’ direction. Pfu DNA Polymerase also exhibits 3’→5’ exonuclease (proofreading) activity that enables the polymerase to correct nucleotide incorporation errors. It has no 5’→3’ exonuclease activity.
The error rate of Pfu DNA Polymerase in PCR is 2.6 x 10-6 errors per nt per cycle as determined by a modified method described by Lundberg et al. (see Reference 2). Accordingly, the accuracy of PCR is 3.8 x 105. Accuracy is an inverse of the error rate and shows an average number of correct nucleotides incorporated before an error occurs. The Pfu DNA Polymerase has no detectable reverse transcriptase activity. dUTP, dITP, and primers containing these nucleotides should not be used in PCR with Pfu DNA Polymerase because the binding of this enzyme to DNA templates with uracil and hypoxanthine stalls DNA synthesis (see References 3,4).
J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, ed. 3, 2001).
K. S. Lundberg, et al., High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1-6 (1991).
G. Shuttleworth et al., Recognition of the pro-mutagenic base uracil by family B DNA polymerases from Archaea. J. Mol. Biol. 337, 621-634 (2004).
P. Gruz et al., Processing of DNA lesions by archaeal DNA polymerases from Sulpholobus solfataricus. Nucleic Acids Res. 31, 4024-4030 (2003).