基于FOK1酶体系的PCR防污染法及在产前诊断中的应用

Controlling carryover PCR contamination based on FOK1 enzyme system and its application in prenatal diagnosis

目的:建立一种有效、实际操作强的聚合酶链反应(polymerase chain reaction,PCR)防污染法并用于产前检测叶酸利用能力。方法:在1条引物的5′端加入限制性内切酶FOK1的识别序列,将FOK1酶加入PCR扩增反应体系中,切断污染的前PCR产物,考察体系中FOK1酶的用量、酶切扩增一体式反应体系成分的比例及污染量的可控程度,并应用于亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MTHFR)基因上C677T突变位点的分析。结果:FOK1酶最佳反应体系却抑制PCR扩增;在TaKaRa反应体系中,0.5 U FOK1酶可完全切割≤0.1μL含有酶切位点的前扩增产物,且成功扩增,而不加入FOK1酶无法控制假阳性结果。FOK1酶切防污染法成功用于MTHFR C677T的测序检测。结论:本方法有效便捷,完全实现了闭管防止PCR污染,不影响后续测序反应,不仅可适用于以PCR扩增为基础的产前诊断等测序分析,更可广泛应用于所有涉及核酸扩增诊断的实验室防治PCR污染中。

Objective:To establish an effective and practical method to solve the problem of polymerase chain reaction(PCR contamination and used it for prenatal diagnosis of folic acid utilization capability.Methods:By adding recognition sequence of a restriction enzyme FOK1 at the 5′end of one PCR primer,carryover PCR contamination was controlled by adding FOK1 enzyme in PCR reaction mixture which contained the contamination from the last PCR products.Effects of the amount of FOK1 enzymatic on amplification efficiency,components in PCR reaction mixture and the most amounts of contamination which can be controlled were investigated.A mutation of C677 T in MTHFR gene was analyzed.Results:A best reaction system provided by FOK1 enzyme specification inhibited PCR amplification.In TaKaRa reaction system,0.5 U of FOK1 enzyme could completely control the contamination of less than 0.1μL of last amplification products and did not affect the amplification.Otherwise,false positive results were obtained by adding the contamination without FOK1 enzyme.The developed method was successfully applied to the analysis of MTHFR C677 T by sequencing.Conclusion:This method is effective and convenient.The reactions of enzyme digestion and amplification were completed in an unclosed tube.Subsequent sequencing reaction is not affected by the method.The method can not only be used in prenatal diagnosis based on PCR amplification,but also widely applied to control PCR amplification in all the fields involved in nucleic acid amplification.