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){middle dot}C, C{middle dot}A, G{middle dot}T, and T{middle dot}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{middle dot}C and T{middle dot}T, about 10-6 for A{middle dot}A, and less than 10-6 for G{middle dot}A, A{middle dot}G, G{middle dot}G and C{middle dot}C. The transversion mispair C(primer){middle dot}T was extended with high efficiency, about 10-2 compared to a correct A{middle dot}T basepair. The unexpected ease of extending the C{middle dot}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{middle dot}T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myelobiastosis reverse transcriptase and HIV-1 reverse transcrlptase 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.