Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography
Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography
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DOI：
10.1093/nar/30.3.e13
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发表时间：
2002-02-01
影响因子：
14.9
通讯作者：
Lu, YY
中科院分区：
文献类型：
--
作者：
Deng, DJ;Deng, GR;Lu, YY
我们在这里报告了一种新的方法，同时检测CpG甲基化和单核苷酸多态性（SNPs）使用变性高效液相色谱（DHPLC）。使用DHPLC分离亚硫酸氢盐修饰的CpG岛的PCR产物。在确认研究中使用BstUI消化和DNA测序。与BstUI消化测定一致，修饰的hMLH 1启动子的294 bp PCR产物在甲基化细胞系（RKO和Cla，6.7 min）和未甲基化细胞系（PACM 82和MGC 803，6.2 min）之间显示不同的保留时间。13例胃癌及其癌旁正常组织中均未发现hMLH 1甲基化。在胃癌患者的癌组织和正常组织中均检测到一个hMLH 1 SNP。DNA测序结果显示，该SNP在hMLH 1启动子的-93 nt处为G → A变异。在AGS、HEK 293和MKN 45细胞系的考克斯-2启动子的605 bp PCR产物中，DHPLC也获得了双峰色谱图。亚硫酸氢盐处理后，在考克斯-2-甲基化AGS细胞系的色谱图上观察到对应于甲基化CpG岛的另一个峰。总之，亚硫酸氢盐-DHPLC可以快速可靠地检测同源等位基因和异源等位基因CpG岛的甲基化。也可以同时检测靶序列中的SNP。
We report here a novel method to simultaneously detect CpG methylation and single nucleotide polymorphisms (SNPs) using denaturing high performance liquid chromatography (DHPLC). PCR products of bisulfite-modified CpG islands were separated using DHPLC. BstUI digestion and DNA sequencing were used in confirmation studies. Consistent with the BstUI digestion assay, the 294 bp PCR product of the modified hMLH1 promoter showed different retention times between the methylated cell lines (RKO and Cla, 6.7 min) and the unmethylated cell lines (PACM82 and MGC803, 6.2 min). No hMLH1 methylation was observed in 13 primary gastric carcinomas and their matched normal tissues. One hMLH1 SNP was detected in gastric cancer patients, in both cancer and normal tissues. DNA sequencing revealed that the SNP is a G-->A variation at -93 nt of the hMLH1 promoter. A two-peak chromatogram was also obtained in the 605 bp PCR product of the Cox-2 promoter of the AGS, HEK293 and MKN45 cell lines by DHPLC. Another peak corresponding to methylated CpG islands was observed on the chromatogram of the Cox-2-methylated AGS cell line after bisulfite treatment. In conclusion, methylation in homoallelic and heteroallelic CpG islands could be detected rapidly and reliably by bisulfite-DHPLC. A SNP in the target sequence could also be detected at the same time.