Effects of primer-template mismatches on the polymerase chain reaction: Human immunodeficiency virus type 1 model studies

TLDR

We investigated the effects of various primer‑template mismatches on DNA amplification of an HIV‑1 gag region by polymerase chain reaction (PCR). The study examined how different mismatches influence PCR amplification efficiency using standard protocols on the gag region. Single internal mismatches did not affect PCR yield, whereas 3′‑terminal mismatches varied: A:G, G:A, C:C reduced yield ~100‑fold, A:A ~20‑fold, most other 3′ mismatches had little effect, but double mismatches at the last four bases generally lowered yield dramatically, except mismatched T at the 3′‑terminus (even with an adjacent mismatch or two mismatched Ts) still allowed efficient amplification.

Abstract

We investigated the effects of various primer-template mismatches on DNA amplification of an HIV-1 gag region by the polymerase chain reaction (PCR). Single internal mismatches had no significant effect on PCR product yield while those at the 3′-terminal base had varied effects. A:G, G:A, and C:C mismatches reduced overall PCR product yield about 100-fold, A:A mismatches about 20-fold. All other 3′-terminal mismatches were efflcientiy amplified, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches. It shouid be noted that mismatches of T with either G, C, or T had a minimal effect on PCR product yield. Double mismatches within the last four bases of a primer-template duplex where one of the mismatches is at the 3′ terminal nucleotide, in general, reduced PCR product yield dramatically. The presence of a mismatched T at the 3′-terminus, however, ailowed significant amplification even when coupled with an adjacent mismatch. Furthermore, even two mismatched Ta at the 3′-terminus allowed efficient ampiification.