变性高效液相色谱法检测CpG岛胞嘧啶甲基化

目的 建立一种新型的ＣpＧ岛胞嘧啶甲基化快速检测方法。方法 用亚硫酸氢钠处理ＤＮＡ，丙用链特异性聚合酶链反应（ＰＣＲ）对错配修复基因hＭＬＨ１启动子含ＣpＧ位点的靶序列进行扩增；利用变性高效液相色谱法（ＤＨＰＬＣ）在部分变性温度下测定靶序列的保留时间，并与亚硫酸氢钠－酶切法测定结果进行比较。结果 用ＤＨＰＬＣ对结肠癌细胞株ＲＫＯ和胃癌细胞株ＰＡＣＭ８２的hＭＬＨ１启动子进行测定，发现ＲＫＯ细胞ＰＣＲ产物的保留时间显长于ＰＡＣＭ８２细胞ＰＣＲ产物的保留时间（６．７min比６．２min）。ＲＫＯ细胞ＰＣＲ产物保留时间的延长是亚硫酸氢钠处理后的模板中胞嘧啶和鸟嘌呤含量较ＰＡＣＭ８２高所致。从此结果分析，可以判断出ＲＫＯ的hＭＬＨ１启动子已甲基化，而ＰＡＣＭ８２细胞未甲基化。此结果与酶切法结果完全一致。结论 新方法可以快速检测ＣpＧ岛胞嘧啶甲基化。

Analysis of the methylation in CpG island by denaturing high-performance liquid chromatography

Objective To establish a new approach for analyzing methylation circumventing the limitations of the existing methods. Methods A region containing CpG sites in mismatch repair gene hMLH1 promoter was amplified by strand-specific polymerase chain reaction (PCR) after sodium bisulfite treatment. The retention time of the PCR product at partially denaturing temperature was determined by denaturing high-performance liquid chromatography (DHPLC). The methylation status obtained by DHPLC was proved by comparing it with that from sodium bisulfite-restriction enzyme digestion. Results Methylation of hMLH1 promoter from a colorectal cancer cell line RKO and a gastric cancer cell line PACM82 was analyzed by DHPLC. The retention time of the PCR product from RKO was obviously longer than that from PACM82 (6.7 min vs. 6.2 min). Thus, it was concluded that the hMLH1 promoter from RKO was methylated, while that from PACM82 was not, since the longer retaining time of RKO was due to the higher C/G content after sodium bisulfite treatment. The results from DHPLC were consistent with those from sodium bisulfite-restriction enzyme digestion. Conclusion DHPLC method is a rapid and reliable approach for analyzing methylation in CpG island of the sequence of interest.