The Effect of Single Mismatches on Primer Extension

Abstract

Background: Allele-specific PCR is an important diagnostic tool that identifies single-nucleotide variants by preferential amplification of a particular allele, using primers that are mismatched to all but one allele variant. Methods: We applied a fluorescent stopped-flow polymerase assay to measure extension rates from oligonucleotide hairpins to simulate primer–template pairs. Under PCR-applicable conditions, reaction rates were recorded in nucleotides per second per polymerase (nt/s/poly). The effects of temperature, potassium chloride, mismatch type, and position were studied with primarily a deletion mutant of Thermus aquaticus (Taq) DNA polymerase and 135 oligonucleotide sequences. Results: Rates at 65 °C were between 205 ± 11 and 177 ± 8 nt/s/poly for matched templates and between 4.55 ± 0.21 and .008 ± 0.005 nt/s/poly for 3′-mismatched templates. Although extension rates progressively increased with mismatches further away from the 3′ end, rates were still reduced by as much as 84% with a C · C mismatch 6 bases from the 3′ end. The optimal extension temperature for matched sequences was 70°C, shifting to 55–60°C for 3′ mismatches. KCl inhibited mismatch extension. The Michaelis constant (K_m) was increased and the unimolecular rate constant (k_cat) decreased for 3′ mismatches relative to matched templates. Conclusions: Although primer extension of mismatches depends on mismatch type and position, variation also depends on local sequence, KCl concentration, and the type of polymerase. Introduction of 3′ mismatches reduces the optimal temperature for extension, suggesting higher annealing temperatures for better allele discrimination. Quantitative descriptions of expected specificity in allele-specific PCR provide additional design direction and suggest when other methods (e.g., high-resolution melting analysis) may be a better choice.