Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR.
Thermus aquaticus (Taq) DNA polymerase was used to measure the extension efficiency for all configurations of matched and mismatched base pairs at template-primer 3'-termini. The transition mispairs, A(primer).C, C.A, G.T, and T.G were extended 10(-3) to 10(-4)-fold less efficiently than their correctly paired counterparts. Relative efficiencies for extending transversion mispairs were 10(-4) to 10(-5) for T.C and T.T, about 10(-6) for A.A, and less than 10(-6) for G.A, A.G, G.G and C.C. The transversion mispair C(primer).T was extended with high efficiency, about 10(-2) compared to a correct A.T basepair. The unexpected ease of extending the C.T mismatch was not likely to have been caused by primer-template misalignment. Taq polymerase was observed to bind with similar affinities to each of the correctly paired and mispaired primer-template 3'-ends. Thus, the failure of Taq polymerase to extend mismatches efficiently appears to be an intrinsic property of the enzyme and not due to an inability to bind to 3'-terminal mispairs. For almost all of the mispairs, C.T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myeloblastosis reverse transcriptase and HIV-1 reverse transcriptase which extend most mismatched basepairs permissively. Relative mismatch extension efficiencies for Taq polymerase were measured at 45 degrees C, 55 degrees C and 70 degrees C and found to be independent of temperature. The mispair extension data should be important in designing experiments using PCR to distinguish between sequences that vary by a single nucleotide.
Huang
M M
MM
Department of Biological Sciences, University of Southern California, Los Angeles 90089-1340.
Arnheim
N
N
Goodman
M F
MF
D001482:Base Composition / Q000235:genetics*; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D019914:Taq Polymerase; D013696:Temperature
D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase