Kwok S, Kellogg D E, McKinney N, Spasic D, Goda L, Levenson C, Sninsky J J
Department of Infectious Diseases, Cetus Corporation, Emeryville, CA.
Nucleic Acids Res. 1990 Feb 25;18(4):999-1005. doi: 10.1093/nar/18.4.999.
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.
我们研究了各种引物-模板错配对聚合酶链反应（PCR）扩增HIV-1 gag区域DNA的影响。单个内部错配对PCR产物产量没有显著影响，而3'-末端碱基处的错配则有不同影响。A:G、G:A和C:C错配使总体PCR产物产量降低约100倍，A:A错配降低约20倍。所有其他3'-末端错配均能有效扩增，尽管G:G错配似乎比其他错配更易受序列背景和dNTP浓度的影响。应当指出的是，T与G、C或T的错配对PCR产物产量的影响最小。引物-模板双链体最后四个碱基内的双重错配，其中一个错配位于3'末端核苷酸处，通常会显著降低PCR产物产量。然而，即使3'-末端存在错配的T，即便与相邻错配同时存在，也能实现显著扩增。此外，即使3'-末端有两个错配的T也能实现有效扩增。
