Shi Xianzhe, Li Jianhua, Zhao Chunxia, Lv Shen, Xu Guowang
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, People's Republic of China.
Biomed Chromatogr. 2006 Aug;20(8):815-20. doi: 10.1002/bmc.606.
DNA methylation is an important epigenetic modification that alters transcription in those genes containing CpG islands. In this report, a novel DNA methylation analysis method was developed employing bisulfite-single strand conformation polymorphism combined with capillary electrophoresis (bisulfite-SSCP-CE). During the bisulfite treatment of genomic DNA, a high concentration of sodium bisulfite (4.8 mol/L) was preferred in order to shorten reaction time and minimize template degradation. The methylated and unmethylated ssDNA of hMLH1 promoter were simultaneously separated under the optimized CE conditions, including 6% SLPA with 10% glycerol as sieving medium, 25 degrees C as separation temperature and 12 kV as running voltage. The heterogeneous methylation of hMLH1 promoter was identified in 13 of 64 colorectal cancer patients. Moreover, hMLH1 promoter methylation had a significant relationship with protein expression loss and increased with the age of patients. Our results indicated that DNA methylation analysis for a large number of clinical samples would be facilitated by use of the bisulfite-SSCP-CE method.
DNA甲基化是一种重要的表观遗传修饰，可改变那些含有CpG岛的基因的转录。在本报告中，开发了一种新的DNA甲基化分析方法，该方法采用亚硫酸氢盐-单链构象多态性结合毛细管电泳（亚硫酸氢盐-SSCP-CE）。在对基因组DNA进行亚硫酸氢盐处理时，为了缩短反应时间并使模板降解最小化，优选高浓度的亚硫酸氢钠（4.8 mol/L）。在优化的CE条件下，包括以6% SLPA和10%甘油作为筛分介质、25℃作为分离温度以及12 kV作为运行电压，hMLH1启动子的甲基化和未甲基化单链DNA可同时分离。在64例结直肠癌患者中的13例中鉴定出hMLH1启动子的异质性甲基化。此外，hMLH1启动子甲基化与蛋白表达缺失显著相关，且随患者年龄增加而增加。我们的结果表明，使用亚硫酸氢盐-SSCP-CE方法将有助于对大量临床样本进行DNA甲基化分析。
