Nucleic Acids Research, 1990, Vol. 18, No. 4 999-1005
© 1990
MOLECULAR BIOLOGY |
Effects of primer-template mismatches on the polymerase chain reaction: Human immunodeficiency virus type 1 model studies
Department of Infectious Diseases Cetus Corporation, Emeryville, CA, USA 1Department of Chemistry Cetus Corporation, Emeryville, CA, USA
Received September 18, 1989. Revised January 19, 1990. Accepted January 19, 1990.
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.
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