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Pfu DNA Polymerase

Not available in the USA


Highly thermostable DNA polymerase for PCR applications requiring high fidelity.
  
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Amplification of DNA fragments of various lengths using Pfu DNA Polymerase

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.

Highlights

  • Eight times more accurate than Taq DNA polymerase
  • Highly thermostable – remains 95% active after 2 hours incubation at 95°C
  • Generates blunt-end PCR products
  • Incorporates modified nucleotides (e.g., biotin-, digoxigenin-, fluorescently-labeled nucleotides)
  • Increased PCR yields with native Pfu DNA Polymerase

Applications

  • High fidelity PCR
  • Generation of PCR products for cloning and expression
  • RT-PCR for cDNA cloning and expression
  • Blunt-end PCR cloning (see Reference 1)

Includes

  • Pfu DNA Polymerase (native or recombinant, 2.5 U/µL)
  • 10X Pfu Buffer with MgSO4
  • 10X Pfu Buffer
  • 25 mM MgSO4

Note

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).

  
Storage Condition-20 C
HazardousNo
10X Pfu Buffer200 mM Tris-HCl (pH 8.8 at 25°C), 100 mM (NH4)2SO4, 100 M KCl, 1% (v/v) Triton X-100, 1 mg/mL BSA.
10X Pfu Buffer with 20 mM MgSO4200 mM Tris-HCl (pH 8.8 at 25°C), 100 mM (NH4)2SO4, 100 mM KCl, 1 mg/mL BSA, 1% (v/v) Triton X-100, 20 mM MgSO4.
Definition of Activity Unit
  • One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 minutes at 72°C.
  • Enzyme activity is assayed in the following mixture: 20 mM Tris-HCl (pH 8.8 at 25°C), 2 mM MgSO4, 10 mM (NH4)2SO4, 10 mM KCl, 0.1 mg/mL BSA, 0.1% (v/v) Triton X-100, 0.75 mM activated calf thymus DNA, 0.2 mM of each dNTP, 0.4 MBq/mL [3H]-dTTP.
InactivationInactivated by phenol/chloroform extraction.
Molecular Weight90 kDa monomer
Quality Control
  • The absence of endo- and exodeoxyribonucleases confirmed by appropriate quality tests.
  • Functionally tested in PCR.
SourcePfu DNA Polymerase (native) Pyrococcus furiosus. Pfu DNA Polymerase (recombinant) E.coli with a cloned pol gene from Pyrococcus furiosus.
Storage Buffer20 mM Tris-HCl (pH 8.2), 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, 0.1% (v/v) Nonidet P40, 0.1% (v/v) Tween 20, and 50% (v/v) glycerol.

References

  1. J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, ed. 3, 2001). 

  2. K. S. Lundberg, et al., High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1-6 (1991).

  3. 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).

  4. P. Gruz et al., Processing of DNA lesions by archaeal DNA polymerases from Sulpholobus solfataricus. Nucleic Acids Res. 31, 4024-4030 (2003).

Product Guides and Selectors

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