Discrimination of primer 3'-nucleotide mismatch by taq DNA polymerase during polymerase chain reaction. Academic Article uri icon

abstract

  • 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.

published proceedings

  • Anal Biochem

altmetric score

  • 6

author list (cited authors)

  • Ayyadevara, S., Thaden, J. J., & Shmookler Reis, R. J.

citation count

  • 143

complete list of authors

  • Ayyadevara, S||Thaden, JJ||Shmookler Reis, RJ

publication date

  • August 2000