title: 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.
authors.affiliation: Division of Research and Development, Stratagene, Inc., La Jolla, CA 92037.
authors.forename: K S
authors.initials: KS
authors.lastname: Lundberg
authors.forename: D D
authors.initials: DD
authors.lastname: Shoemaker
authors.forename: M W
authors.initials: MW
authors.lastname: Adams
authors.forename: J M
authors.initials: JM
authors.lastname: Short
authors.forename: J A
authors.initials: JA
authors.lastname: Sorge
authors.forename: E J
authors.initials: EJ
authors.lastname: Mathur
mesh_terms: D001105:Archaea / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
chemical_list: C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase