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.

Myoglobin as a polymerase chain reaction (PCR) inhibitor: a limitation for PCR from skeletal muscle tissue avoided by the use of Thermus thermophilus polymerase.

Skeletal muscle tissue contains polymerase chain reaction (PCR) inhibitors that are coextracted by conventional nucleic acid extraction procedures. Myoglobin, a heme-containing molecule, was shown to act as a potent Thermus aquaticus DNA polymerase inhibitor and is likely to be involved in muscle tissue-associated PCR inhibition. The use of Thermus thermophilus DNA polymerase avoids muscle tissue-associated PCR inhibition, and should be used in case of small amounts or instability of the targeted nucleic acid.