摘要
目的建立准确测定肿瘤组织中SNCG基因CpG岛甲基化拷贝的含量的方法.方法利用亚硫酸氢钠修饰-克隆测序法确定胃组织该CpG岛中关键性CpG位点,然后利用限制性内切酶建立了测定该位点甲基化状态的分析法(COBRA),再利用变性高效液相色谱技术进行定量.结果对2个肿瘤细胞系、2个正常人胃黏膜样品、2对原发性胃癌及其癌旁非癌组织进行分析发现,在该CpG岛的16个CpG位点中,-88位等5个CpG位点的甲基化状态与整个CpG岛的甲基化状态一致;利用限制性内切酶Acil能够区分该位点甲基化状态:在该位点未甲基化时(GTGG)不能酶切,而甲基化时(GCGG)酶切敏感;在变性高效液相色谱仪上甲基化片段和非甲基化片段能够完全分离;根据色谱峰面积可知两者的比例,在1.25%~100%范围内有良好的线性关系,重现性较好;对克隆测序样品进行限制性内切酶-变性高效液相色谱测定,结果与克隆测序一致.结论建立的限制性内切酶-变性高效液相色谱法能够定量测定SNCG基因CpG岛甲基化.
Objective To setup a quantitative assay for detection of methylation of SNCG CpC island in human tissue samples.Methods Methylation status of the 16 tested CpG sites within the CpG island was analyzed by bisulfite clone-sequencing for 2 gastric carcinoma cell lines,2 normal gastric mucosa samples,and 2 pairs of primary gastric carcinomas and their corresponding non-neoplastic tssues,respectively.Results The methylation of-88 and other four CpC sites was well correlated with the"methyaion of the overall CpG island.Thus,a combined bislte-restiction assaey(COBRA)was developed based on the enzyme Acil,which digested the only one GCGC sequence in the PCR products of the methylated CpG island,but not the GTGG in the demethylated one.The digested fragments(144 bp and 85 bp)and undigested fragment(229 bp)could be completely separated by denaturing high perfomance liquid chromatography(DHPLC).According to the peak areas of these fragments,the proportion of the methylated copies of the SNCG CpG island was calculated easily.The result of the COBRA-DHPLC assay was reprodueible and consistent with that of clone-sequencing Conclusion A COBRA-DHPLC assay is setup scesfully for quantifcation of methylation of the SNCG CpG island.
作者
周静
文贤子
邓大君
ZHOU Jing;WEN Xian-zi;DENG Da-jun(Peking University School of Oncology and Beijing Instiute for Cancer Research,100036 Bejing,China)
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2007年第S01期20-24,共5页
Chinese Journal of Preventive Medicine
基金
国家"973"计划项目资助(2005CB522403)
关键词
CPG岛
甲基化
色谱法
高效液相
CpG island
Methylation
Chromatography,high pressure liquid
