PCR-ACRS方法筛查CYP21基因P459H的突变位点

目的：研究21-羟化酶基因（CYP21）第10外显子P459H突变（CCC→CAC）在正常人群中的发生率，分析P459H是点突变还是基因多态。方法:设计特异性引物扩增CYP21基因3-10外显子，PstⅠ限制性内切酶消化验证PCR产物为特异性扩增，在此基础上，应用PCR结合扩增引进限制性酶切位点方法(PCR-ACRS)扩增首次PCR产物，FspⅠ限制性内切酶酶切后用10%聚丙烯酰胺凝胶电泳筛查第10外显子P459H的发生率。 结果:100例正常人CYP21基因第10外显子密码子459均为CCC，即均未出现P459H变异。 结论:CYP21基因P459H为点突变，排除基因多态的可能；另外，PCR-ACRS是一种快速、可靠的检测基因点突变的有效途径。

Screening of CYP21 gene P459H mutation by PCR-ACRS

AIM:To investigate the frequency of P459H (CCC→CAC) in exon 10 of CYP21 gene among normal population. METHODS:The exons 3-10 of CYP21 gene were amplified with polymerase chain reaction (PCR). The PCR round products were digested by restriction enzyme Pst I to confirm that CYP21 gene was specifically amplified. PCR-based amplification-created restriction site (PCR-ACRS) was performed using the first round PCR products as template. After the second PCR products were digested by restriction enzyme Fsp I, 10% polyacrylamide gel electrophoresis was used to screen the frequency of P459H in exon 10. RESULTS:The codon 459 in exon 10 of CYP21 gene was all CCC among 100 normal cases tested. CONCLUSION:P459H (CCC →CAC) of CYP21 gene might be a novel point mutation causing CAH. Furthermore, PCR-ACRS was a fast and safe method for gene mutation screening.