过量DMSO显著降低低模板浓度PCR扩增的特异性

DMSO通常经验性地用于提高PCR扩增的效率,但是过量的DMSO可以显著降低特异序列的扩增效率,尤其是导致非特异性扩增的现象却被忽视.在6%的DMSO存在时,开始出现非特异条带,同时特异扩增产物减少.本文首次报道DMSO对PCR产物特异性的影响并确定了克服上述现象产生的途径,即通过增加模板-引物的比例消除非特异条带.而通常提高复性温度只能部分减少非特异产物,不能避免非特异扩增.本文结果有助于提高常规PCR,尤其是分子遗传学分析中经常使用的随机扩增多态性DNA(randomamplifiedpolymorphicDNA,RAPD)检测的准确度。 Abstract:DMSO is the most often empirically used to increase efficiency of PCR.However,it is ignored that DMSO can also decrease the amplification of specific sequences and result in non- specific amphfications.Non-specific bands are detected at 6% DMSO,as specific amplification declines.This is the first report about the effects of DMSO on the specificity of PCR and establishment of the method for avoiding non-specific products,which can be overcome by increasing template-primer ratio rather than annealing temperature.Our data will be of much help for random amphfied polymorphic DNA (RAPD) assays for molecular genetic analyses.

Excessive DMSO Dramatically Reducing the Specificity of PCR Amplification at Low Concentrations of Template