酶切反应体系对后续PCR特异性的影响.docVIP

基因甲基化PCR检测体系的优化 揣征然，黄 庆，黄君富，府伟灵 第三军医大学西南医院检验科，重庆 400038 摘要目的 优化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 未纯化酶切产物直接进行PCR特异性纯化用PCR缓冲液替代酶切缓冲液后PCR反应特异性。结论 酶切后续PCR反应特异性，PCR缓冲液替代酶切缓冲液能够有效提高后续PCR特异性。 关键词；；基因甲基化R446.9；R446.19 Optimization for PCR Detection System of Gene Methylation Chuai Zhengran，Huang Qing，Huang Junfu， F Weiling (Department of Clinical Laboratory, Southwest Hospital, Third Military Medical University, Chongqing 400038, China) [Abstract] Objective Optimize methylation sensitive restriction enzyme (MSRE) PCR detection system. Methods Methylation of RASSF1A gene from 10 healthy non-pregnant women was detected by MSRE-PCR. Results The specificity of PCR with purified digested products or with PCR buffer is higher, compared with non-purified digested products. Conclusion The digestion reaction 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. PCR buffer is recommended for both digestion and PCR due to convenience. [Keywords] endonuclease reaction buffer; PCR buffer; Purification; Gene methylation detection Supported by the General Program of National Natural Science Foundation of China; Corresponding author: Fu weiling, Tel: (023) Email:weilingfu@ DNA甲基化是指在DNA甲基转移酶的作用下，基因DNA序列CpG双核苷酸的胞嘧啶5’碳位共价键结合一个甲基基团[1]DNA甲基化基因功能的重要修饰手段恶性肿瘤、感染性疾病、遗传性疾病及出生缺陷等[2-6]。因此，基因甲基化重大疾病或疗效监测。目前，甲基化敏感限制性内切酶（methylation sensitive restriction enzyme, MSRE）PCR技术[7]基因甲基化最常用的手段之一，本文健康未孕女性的RASSF1A基因甲基化[]水平过程中，初步探索了酶切反应体系对后续PCR的影响，并对进行了。 1 材料和方法 1.1 一般资料 取2013年1月至3月在我院体检中心参加健康体检的10例未孕女性，年龄24-34岁，平均年龄为28岁，入选者签署知情同意书。 1.2主要试剂和仪器 DNA提取采用QIAGENE公司的QIAamp Blood Kit试剂盒；酶切反应在My Gene Series Peltier Thermal Cycler中进行；TF Filter Tips（Purification Filter TrimGen试剂盒）进行；PCR扩增采用BIO-RAD CFX96 TMReal-Time System；BstUI购自New England Biolabs公司；Ex Taq HS DNA Polymerase试剂盒、DL2000 Marker购于大连宝生物有限公司；PicoGreen dsDNA Assay Kit