PCR扩增3因素对低拷贝模板STR分型的影响研究

目的探讨低拷贝模板(low copy number,LCN)STR扩增方法,提高LCN检材的检验成功率。方法采用Profiler P lusTM试剂盒与9947A对照DNA,改变Taq酶量、体系、循环次数3个因素进行扩增检验,了解各变量对扩增检测的影响。结果对低拷贝模板DNA,单纯增加Taq酶量或反应体系,扩增效率改善不明显;增加循环数,显著提高检验灵敏度;低于0.01ng的模板DNA,同时增加扩增体系、Taq酶量、循环数在一定程度上提高扩增效率。结论对于影响扩增的Taq酶量、体系、循环次数3个因素中,循环数影响最大,但应慎用34次及以上循环数;三者同时增加,对于低于0.01ng模板DNA的扩增可有效改善。

A study on the influence of three factors in PCR to LCN-STR genotyping

Objective To explore the methods to amplify LCN-STR,and improve the success rate of LCN-STR typing.Methods Using the Profiler Plus~(TM) kits and 9947A control DNA,the effects of three factors in PCR on the results of the STR amplification were observed through changing the amount of Taq enzyme,reaction volume and PCR cycles.Results For LCN template DNA,if only increased the amount of Taq enzyme or the reaction volume,the results of STR amplification were scarcely improved,but if increased PCR cycles,the detection sensitivity were significantly improved.For template DNA below 0.01ng,when the amount of Taq enzyme,reaction volume and PCR cycles were increased at the same time,the amplification efficiency could be improved in a certain extent.Conclusion Among the three factors in PCR including amount of Taq enzyme,reaction volume and PCR cycles,the cycles was the most important,but beyond 34 cycles should be used carefully.Increasing the amount of Taq enzyme,reaction volume and PCR cycles simultaneously,better results could be obtained for template DNA below 0.01ng.