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' nucleotide, Thermus aquaticus (Taq) DNA polymerase was highly specific for template complementarity to this base, but was somewhat less constrained opposite the penultimate nucleotide. In contrast, primers with a 3'-terminal A were less efficiently amplified regardless of the corresponding nucleotide on the template strand. Thus, allele-specific PCR with Taq polymerase offers the greatest template discrimination (40- to 100-fold) against mismatch to a primer's 3'-terminal T, G, or C, but not A. Nucleotides at the penultimate position are responsible for roughly one-fifth as much mismatch discrimination (8- to 20-fold), and amplification efficiency is reduced when T and especially A occupy this primer position. We thus have defined conditions which allow robust discrimination for PCR-mediated analysis of single-nucleotide polymorphisms (SNPs), and for reduction in complexity of anchor-ligation PCR products.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
S
S
Ayyadevara
J J
JJ
Thaden
R J
RJ
Shmookler Reis
D000818:Animals; D020137:Base Pair Mismatch; D017173:Caenorhabditis elegans / Q000235:genetics; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000494:pharmacology*; D013698:Templates, Genetic
D017931:DNA Primers; D004247:DNA; D019914:Taq Polymerase