PyroMarkQ24焦磷酸测序法测定DNA甲基化张濛1朱德广2唐德良.docVIP

PyroMark Q24焦磷酸测序法测定DNA甲基化 张 濛1 朱德广 2 唐德良* 2 张丁*1 （1.河南省疾病预防控制中心，450016，河南，中国 美国哥伦比亚大学公共卫生学院，10032，纽约，美国） [摘要]目的：建立一种基于亚硫酸氢钠转化后DNA进行特异性PCR,结合PyroMark Q24进行焦磷酸测序来测定DNA甲基化状态的方法。方法：选取人外周血提取DNA样本7份，经亚硫酸氢钠转化后，使用BDNF基因待测片段的前端引物和后端引物进行甲基化特异性的PCR，最后采用PyroMark Q24焦磷酸测序系统用于甲基化etermination of DNA methylation by pyrosequencing with PyroMark Q24 Zhang Meng Zhu Deguang Tang Deliang Zhang Ding Henan Center for Disease Control and Prevention, 450016, Henan,China; 2. Mailman School of Public Health of Columbia University, New York, 10032, USA) [Abstract]Objective: Establish a new method to determine DNA methylation by pyrosequencing with PyroMark Q24, which is based on specific PCR after DNA being converted by sodium bisulfite. Methods: Extract 7 DNA samples from human peripheral blood, and carry out methylation-specific PCR with forward primer and reverse primer of given BDNF genes after DNA being converted by sodium bisulfite, and finally test and analyse DNA methylation of given BDNF genes by pyrosequencing with PyroMark Q24. Results: Carry out specific PCR with given primers and conditions of BDNF genes, the results show that specific BDNF gene amplified band is 155bp and 7 DNA samples are consistent, the negative control( H2O) has no specific amplified bands. Analyze the results of the BDNF gene methylation status at 4 different sites with PyroMark Q24 software, the negative control( H2O) has no signal response. Conclusion:This method is a quantitative detection methods for DNA methylation that is very promisingly useful and it will play a major role in the study of epigenetics in the future. [Key words] Epigenetics Methlylation Pyrosequence 表观遗传学（Epigenetics）中DNA甲基化是目前国际上的研究热点，此反应主要发生在5′-CG-3′的DNA序列中，即CpG岛甲基化水平的改变。甲基化状态的改变PyroMark Q24焦磷酸测序法测定出DNA的甲基化水平的一套完整成熟的测定方法。 1 材料和方法 1.1 DNA的制备[1,2，3] 样本来源：以外周血为DNA的提取材料，即是从有核的白细胞中提取DNA。静脉抽取血样EDTA抗凝,用等体积的酚抽提一次，离心收集水相，用等体积的（酚氯仿）混合物抽提一次，离心收集水相入0.1倍体积3mol/L乙酸钠(PH5.2)与2倍体积-20预冷无水乙醇。 DNA纯度检测DNA的纯度用260/OD280比值利用PyroMark Q24 设计软件可以对