聚合酶链式反应变性及退火温度与产物假阴性之间的关系

目的探讨聚合酶链式反应(PCR)变性及退火温度与产物假阴性之间的关系。方法以含有HBV完整基因组的重组质粒pUC-19为模板进行PCR扩增,人为改变PCR循环的变性或退火温度,探讨其与产物假阴性之间的关系。结果随着变性温度的降低和退火温度的升高,PCR扩增效率降低,产物量明显减少。当变性温度降低至85℃及85℃以下,退火温度升高至72℃时,琼脂糖凝胶电泳肉眼观察不到扩增产物。结论过低的变性温度和过高的退火温度会导致PCR产物出现假阴性。

Relationship of Temperatures for Denaturalization and Annealing to False Negative PCR Result

Objective To explore the relationship of temperatures for denaturalization and annealing to the false negative PCR result.Methods Amplify pre-C gene of HBV by PCR using recombinant plasmid pUC-19 carrying whole HBV genome as a tem-plate.Change the temperatures for denaturalization and annealing and observe their effect on PCR result.Results Both PCR effica-cy and the yield of PCR product decreased with the decreasing temperature for denaturalization and the increasing temperature for annealing.When the temperature for denaturalization decreased to 85℃ or below,and that for annealing increased to 72℃,no visual PCR product was observed on agarose gel electrophoretic profile.Conclusion False negative PCR result appeared when the temper-ature for denaturalization was too low while that for annealing was too high.