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 efficiently amplified, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches. It should 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, allowed significant amplification even when coupled with an adjacent mismatch. Furthermore, even two mismatched Ts at the 3'-terminus allowed efficient amplification.

Keywords

BiologyPrimer (cosmetics)Polymerase chain reactionDuplex (building)Molecular biologyBase pairPolymeraseDNAContext (archaeology)GeneticsVirologyGeneChemistry

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Publication Info

Year
1990
Type
article
Volume
18
Issue
4
Pages
999-1005
Citations
1044
Access
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Shirley Kwok, D.E. Kellogg, Nancy McKinney et al. (1990). Effects of primer-template mismatches on the polymerase chain reaction: Human immunodeficiency virus type 1 model studies. Nucleic Acids Research , 18 (4) , 999-1005. https://doi.org/10.1093/nar/18.4.999

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DOI
10.1093/nar/18.4.999