Christopherson C, Sninsky J, Kwok S
Roche Molecular Systems Inc., 1145 Atlantic Avenue, Alameda, CA 94501, USA.
Nucleic Acids Res. 1997 Feb 1;25(3):654-8. doi: 10.1093/nar/25.3.654.
We investigated the effects of internal primer-template mismatches on the efficiency of reverse transcription and PCR amplification. As models, RNA transcripts representative of different HIV-1 group M subtypes were evaluated with a previously described gag primer pair system. We observed that the presence of two to four mismatches in the primer-template duplexes did not have a significant effect on RT-PCR. However, the presence of five and six mismatches with the 28 and 30 base primers reduced PCR product yield by approximately 22- and 100-fold respectively, relative to the homologous template. The amount of reduction was reproducible from experiment to experiment and was independent of the initial copy number input. Under the conditions used, viral RNA measurements of the more divergent HIV-1 subtypes (A and E) would be underestimated, while isolates of subtypes B, C, D and F-H are expected to be efficiently amplified and accurately measured. The reduced amplification efficiency for targets similar to HIV subtypes A and E can be improved 4- to 10-fold by lowering the annealing temperature and implementing a reverse transcription step that gradually increases in temperature. The additional substitution of either 5-methylcytosine for cytosine throughout or the substitution of inosine at positions of variable bases resulted in a <4-fold difference in product yield between the homologous and most divergent templates.
我们研究了内部引物-模板错配对逆转录效率和PCR扩增的影响。作为模型，使用先前描述的gag引物对系统评估了代表不同HIV-1 M组亚型的RNA转录本。我们观察到引物-模板双链体中存在两到四个错配，对RT-PCR没有显著影响。然而，与28个碱基和30个碱基的引物存在五个和六个错配时，相对于同源模板，PCR产物产量分别降低了约22倍和100倍。减少的量在每次实验中都是可重复的，并且与初始输入拷贝数无关。在所使用的条件下，HIV-1更具差异的亚型（A和E）的病毒RNA测量值会被低估，而B、C、D和F-H亚型的分离株有望被有效扩增并准确测量。对于与HIV A和E亚型相似的靶标，通过降低退火温度并实施温度逐渐升高的逆转录步骤，扩增效率降低的情况可提高4至10倍。在整个过程中用5-甲基胞嘧啶替代胞嘧啶或在可变碱基位置用次黄嘌呤替代，同源模板和差异最大的模板之间的产物产量差异小于4倍。
