Discrimination of primer 3 '-nucleotide mismatch by Taq DNA polymerase during polymerase chain reaction

We investigated the effect of primer-template mismatch on the efficiency of polymerase chain reaction. For primers with T, C, or G as the 3' nucleotid e, Thermus aquaticus (Taq) DNA polymerase was highly specific for template complementarity to this base, but was somewhat less constrained opposite th e penultimate nucleotide. In contrast, primers with a 3'-terminal A were le ss efficiently amplified regardless of the corresponding nucleotide on the template strand. Thus, allele-specific PCR with Tag polymerase offers the g reatest template discrimination (40- to 100-fold) against mismatch to a pri mer's S'-terminal T, G, or C, but not A. Nucleotides at the penultimate pos ition are responsible for roughly one-fifth as much mismatch discrimination (8- to 20-fold), and amplification efficiency is reduced when T and especi ally A occupy this primer position. We thus have defined conditions which a llow robust discrimination for PCR-mediated analysis of single-nucleotide p olymorphisms (SNPs), and for reduction in complexity of anchor-ligation PCR products. (C) 2000 Academic Press.