扩增引进限制性酶切位点技术检测CYP2C9基因的多态性

Identification of polymorphic CYP2C9 alleles by amplification-created restriction sites

建立了一种简便、快速、准确的检测CYP2C9基因Arg144Cys位点和Ile359Leu位点多态性的新方法.该方法采用与模板具有1个碱基错配的引物通过PCR在扩增产物中引进限制性酶切位点,即扩增引进限制性酶切位点(amp lification-created restriction sites,ACRS),其中通过上游引物在扩增Arg144Cys位点的片断中引入AvaⅡ切点,使得野生型基因CYP2C9*1和突变型基因CYP2C9*2的PCR产物中分别含有2个和1个AvaⅡ切点;通过下游引物在扩增Ile359Leu位点的片断中引入MvaⅠ切点,使得野生型基因CYP2C9*1和突变型基因CYP2C9*3的PCR产物中分别含有1个和2个MvaⅠ切点.将来自Arg144Cys位点和Ile359Leu位点的PCR产物各取一份测序,表明错配碱基成功引入PCR产物中.将包含Arg144Cys位点的PCR产物用AvaⅡ消化,将包含Ile359Leu位点的PCR产物用MvaⅠ消化,然后通过聚丙烯酰胺凝胶电泳-银染进行基因分型.由于野生型基因和突变型基因的PCR产物中都至少含有1个酶切位点,能完全避免因限制性核酸内切酶失活或酶切不完全而导致的基因分型错误.

A simple, rapid and accurate method for identifying the CYP2C9 aUelic variants was established. The PCR -based amplification -created restriction site was performed using one base mismatch primers at 3＇ - end. An Ava Ⅱ recognition site was introduced into the PCR products spanning Arg144Cys Loci , the PCR products originating from wild and mutant alleles contain two and one Ava Ⅱ recognition site respectively ; A Mva Ⅰ recognition site was introduced into the PCR products spanning Ile359Leu Loci , the PCR products originating from wild and mutant alleles contain one and two Mva Ⅰ recognition site respectively, Sequencing of two samples showed that the mismatch bases were introduced into PCR products successfully. Then the PCR products were digested with corresponding restriction enzymes, the fragments were separated by polyacrylamide gel electrophoresis （PAGE）and appeared by silver staining. Because the PCR products originating from wild and mutant alleles contain at least one recognition site of corresponding restriction enzymes,this method can avoid the false genotyping owing to the inactive restriction enzymes or inadequate digestion.