Deng D, Deng G, Lü Y
Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing 100034, China.
Zhonghua Yi Xue Za Zhi. 2001 Feb 10;81(3):158-61.
To establish a new approach for analyzing methylation circumventing the limitations of the existing 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.
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.
DHPLC method is a rapid and reliable approach for analyzing methylation in CpG island of the sequence of interest.
建立一种新的甲基化分析方法，以克服现有方法的局限性。
亚硫酸氢钠处理后，通过链特异性聚合酶链反应（PCR）扩增错配修复基因hMLH1启动子中含CpG位点的区域。采用变性高效液相色谱（DHPLC）测定PCR产物在部分变性温度下的保留时间。通过与亚硫酸氢钠-限制性内切酶消化法的结果进行比较，验证DHPLC获得的甲基化状态。
用DHPLC分析结肠癌细胞系RKO和胃癌细胞系PACM82的hMLH1启动子甲基化情况。RKO的PCR产物保留时间明显长于PACM82（6.7分钟对6.2分钟）。因此，得出结论：RKO的hMLH1启动子发生了甲基化，而PACM82的未甲基化，因为RKO保留时间较长是由于亚硫酸氢钠处理后C/G含量较高。DHPLC的结果与亚硫酸氢钠-限制性内切酶消化法的结果一致。
DHPLC法是分析目标序列CpG岛甲基化的一种快速可靠的方法。
Zotero 插件