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Why select Pfu DNA Polymerase over Taq DNA Polymerase? How should an amplification...

Why select Pfu DNA Polymerase over Taq DNA Polymerase? How should an amplification reaction containing Pfu polymerase be prepared?

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LabFact #8

PCR products created using Taq, Tth or Tfl DNA Polymerase often have a  3´-A overhang and can be cloned using the  pGEM®-T and pGEM®-T-Easy Vector Systems.
 (from #TM042)

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Pfu DNA Polymerase is a thermostable enzyme isolated from Pyrococcus furiosus Vc1 (DSM3638)(1) . In addition to 5´to 3´ DNA polymerase activity, it also possesses 3´ to 5´ exonuclease (proofreading) activity. Pfu DNA Polymerase exhibits the lowest error rate of any thermostable DNA polymerase studied(2) (3) (4) . Consequently, Pfu DNA Polymerase is useful for polymerization reactions requiring high-fidelity synthesis(2) (3) (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 × 105 (4) to 1 × 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 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

  1. Fiala, G. and Setter, K.O. (1986) Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100°C. Arch. Microbiol. 145, 56.
  2. Flaman, J.M. et al. (1994) A rapid PCR fidelity assay. Nucleic Acids. Res. 22, 3259–60.
  3. Andre, P. et al. (1997) Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence. Genome Res. 7, 843–52.
  4. Cline, J., Braman, J.C. and Hogrefe, H.H. (1996) PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucl. Acid Res. 24, 3546–51.
  5. Lundberg, K.S. et al. (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1–6.
  6. Slater, M. et al. (1998) Pfu DNA Polymerase: A high fidelity enzyme for nucleic acid amplification. Promega Notes 68, 7–10.
  7. Skerra, A. (1992) Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. Nucleic Acids Res. 20, 3551.

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