Discrimination of Primer 3′-Nucleotide Mismatch by Taq DNA Polymerase during Polymerase Chain Reaction

✍ Scribed by Srinivas Ayyadevara; John J. Thaden; Robert J. Shmookler Reis

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 127 KB
• Volume: 284
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4635

✦ Synopsis

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.