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Why select Pfu DNA Polymerase over Taq DNA Polymerase? How
should an amplification reaction containing Pfu DNA Polymerase be prepared?
Pfu DNA Polymerase (Cat.# M7741) is a
thermostable enzyme isolated from Pyrococcus furiosus Vc1 (DSM3638) (1). In
addition to 5´-->3´ DNA polymerase activity, it also possesses 3´-->5´
exonuclease (proofreading) activity. Pfu DNA Polymerase exhibits the lowest error
rate of any thermostable DNA polymerase studied (2-4). Consequently, Pfu DNA
Polymerase is useful for polymerization reactions requiring high-fidelity synthesis (2-5).
The accuracy of a polymerase is the average number of nucleotides the polymerase
incorporates before making an error. The error rate is the reciprocal value of accuracy
and is expressed as the mutation rate per duplicated base pair. The accuracy of Pfu
DNA Polymerase has been reported to be 7.7 x 105 (4) to 1 x 106 (6).
The same PCR-based assay has been used to demonstrate that the accuracy of Pfu
DNA Polymerase is approximately 2-fold higher than Vent® DNA polymerase (New
England Biolabs, Inc.), and approximately 6-fold higher than Taq DNA Polymerase (3,4). To achieve high fidelity, the pH of the 10X Tris-based buffer in
amplification reaction must be greater than 8.6 (at 25°C); at pH 8.0, the fidelity of Pfu
DNA Polymerase is not much better than the fidelity of Taq DNA Polymerase. The pH
of the reaction buffer supplied with Promega Pfu DNA Polymerase is 8.8,
which allows for high fidelity. The reaction buffer also includes 2mM MgSO4 for
optimal enzyme activity (when used with 200µM each dNTP). The fidelity of the reaction
can also be maximized by increasing the amount of input template DNA, as well as by
decreasing the cycle number and minimizing the time spent at the denaturation temperature.
Thus, for routine PCR for the simple detection of product, Taq DNA Polymerase or Tfl
DNA Polymerase is appropriate, while for high-fidelity PCR,
such as gene cloning, gene expression or mutation analysis, Pfu DNA Polymerase is
recommended.
It is critical to add the Pfu DNA Polymerase to the amplification reaction
last, particularly following the addition of dNTPs, as the proofreading activity of the
polymerase may degrade the amplification primers resulting in nonspecific amplification
and reduced product yield. To overcome primer degradation, longer primers with maximized
GC-content can be used. The first fifteen 5´ bases have nearly full protection from
degradation, and as such, a good primer length is 20-35 bases. Primers can also be
protected by introducing a single phosphorothioate bond at their 3´ termini (7). The
amplification reactions should be set up on ice and then placed into a thermocycler
preheated to 95°C. An extension time of 2 minutes for every 1kb to be amplified should be
used.
References
- Fiala, G. and Stetter, K.O. (1986) Pyrococcus furiosus sp. nov. represents a
novel genus of marine heterotrophic archaebacteria growing optimally at 100°C. Arch.
Microbiol. 145, 56.
- Flaman, J.M. et al. (1994) A rapid PCR fidelity assay. Nucl. Acids Res.
22, 3259.
- Andre, P. et al. (1997) Fidelity and mutational spectrum of Pfu DNA
polymerase on a human mitochondrial DNA sequence. Genome Res. 7,
843.
- Cline, J., Braman, J.C. and Hogrefe, H.H. (1996) PCR fidelity of Pfu DNA
polymerase and other thermostable DNA polymerases. Nucl. Acids Res. 24,
3546.
- Lundberg, K.S. et al. (1991) High-fidelity amplification using a thermostable
DNA polymerase isolated from Pyrococcus furiosus. Gene 108,
1.
- Slater, M. et al. (1998) Pfu DNA Polymerase: A High Fidelity Enzyme
for Nucleic Acid Amplification. Promega
Notes 68, 7.
- Skerra, A. (1992) Phosphorothioate primers improve the amplification of DNA sequences by
DNA polymerases with proofreading activity. Nucl. Acids Res. 20,
3551.
Vent is a registered trademark of New England Biolabs, Inc.
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