High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase.
We demonstrate that despite lacking a 3'----5' proofreading exonuclease, the Thermus aquaticus (Taq) DNA polymerase can catalyze highly accurate DNA synthesis in vitro. Under defined reaction conditions, the error rate per nucleotide polymerized at 70 degrees C can be as low as 10(-5) for base substitution errors and 10(-6) for frameshift errors. The frequency of mutations produced during a single round of DNA synthesis of the lac Z alpha gene by Taq polymerase responds to changes in dNTP concentration, pH, and the concentration of MgCl2 relative to the total concentration of deoxynucleotide triphosphates present in the reaction. Both base substitution and frameshift error rates of less than 1/100,000 were observed at pH 5-6 (70 degrees C) or when MgCl2 and deoxynucleotide triphosphates were present at equimolar concentrations. These high fidelity reaction conditions for DNA synthesis by the Taq polymerase may be useful for specialized uses of DNA amplified by the polymerase chain reaction.
Eckert
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Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Kunkel
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TA
D001482:Base Composition; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000494:pharmacology; D009154:Mutation; D016133:Polymerase Chain Reaction; D019914:Taq Polymerase; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D015636:Magnesium Chloride; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase