基因甲基化聚合酶链反应检测体系的优化  

Optimization for PCR detection system of gene methylation

作　　者：揣征然[1] 黄庆[1] 黄君富[1] 府伟灵[1] 

机构地区：[1]第三军医大学西南医院检验科,重庆400038

出　　处：《检验医学与临床》2014年第21期2945-2946,2949,共3页Laboratory Medicine and Clinic

基　　金：国家863重大专项基金资助项目(2011AA02A121;213AA020204);国家自然科学基金资助项目(81071429)

摘　　要：目的优化甲基化敏感限制性内切酶的聚合酶链反应(PCR)检测体系。方法用甲基化敏感限制性内切酶-PCR技术对10例健康未孕女性的RASSF1A基因进行甲基化检测,并对反应的缓冲体系进行优化。结果未经纯化的酶切产物直接进行PCR时,PCR反应特异性降低,出现众多非特异PCR产物;纯化酶切产物或用PCR缓冲液替代酶切缓冲液后,PCR反应特异性变高。结论酶切缓冲液可影响后续PCR反应的特异性,采取纯化措施或以PCR缓冲液替代酶切缓冲液能够有效提高后续PCR特异性,首选PCR缓冲液替代更为简便。Objective To optimize the methylation sensitive restriction enzyme（MSRE）polymerase chain reaction（PCR）detection system.Methods Methylation of RASSF1 Agene from 10 healthy non-pregnant women was detected by MSRE-PCR.The buffer system of reaction was optimized.Results In directly conducting PCR with nonpurified digested products,the specificity of PCR was decreased and many non-specificity PCR products appeared;by purifying digested products or using PCR buffer instead of digested buffer,the specificity of PCR was increased.Conclusion The digested buffer could affect the specificity of subsequent PCR.The purification of digested products or the replacement of digested buffer with PCR buffer can effectively improve the specificity of following PCR.In both,PCR buffer is first choice due to its more convenient.

关 键 词：限制性内切酶缓冲液 聚合酶链反应缓冲液 纯化 基因甲基化检测 

分 类 号：R446.5[医药卫生—诊断学]