High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus.

Abstract:

A thermostable DNA polymerase which possesses an associated 3'-to-5' exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10% of the number of mutations obtained from similar amplifications performed with Taq DNA polymerase. The PCR fidelity assay is based on the amplification and cloning of lacI, lacO and lacZ alpha gene sequences (lacIOZ alpha) using either Pfu or Taq DNA polymerase. Certain mutations within the lacI gene inactivate the Lac repressor protein and permit the expression of beta Gal. When plated on a chromogenic substrate, these LacI- mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the 182 known detectable sites of the lacI gene was 1.6 x 10(-6) for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x 10(-5) error rate for Taq DNA polymerase, after approx. 10(5)-fold amplification.

Polymerases:

Pfu

Topics:

Historical Protein Properties (MW, pI, ...), Fidelity, Nucleotide Incorporation, Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Pfu Lundberg KS1991 3-5' Exonuclease (proofreading) Yes
Pfu Lundberg KS1991 Cloned or native Native organism
Pfu Lundberg KS1991 3-5' Exo Specific Activity 9200 units/mg
Pfu Lundberg KS1991 Tagged No
Pfu Lundberg KS1991 Nucleotide Substitution Rate 1.6E-06 errors/bp
Pfu Lundberg KS1991 Full length or truncated Full length
Pfu Lundberg KS1991 Specific Activity 3.171E+04 units/mg

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