Determination of the heterogeneity of DNA methylation by combined bisulfite restriction analysis and capillary electrophoresis with laser-induced fluorescence

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• 作者：Hua-Chien Chen^a ; Yu-Sun Chang^b ; Shu-Jen Chen^a ; ^{sjchen@cgu.edu.tw} ; Po-Ling Chang^c ; ^{poling@thu.edu.tw}
• 关键词：Capillary electrophoresis ; Laser-induced fluorescence ; DNA methylation ; Separation ; Poly(ethylene) oxide
• 刊名：Journal of Chromatography A
• 出版年：2012
• 期刊代码：47_00219673
• 类别：ch
• 出版时间：23 March, 2012
• 卷：1230
• 期：Complete
• 页码：123-129
• 文件大小：872 K

摘要

The methylation of the promoter region of DNA is an important regulatory mechanism for the downstream gene expression, and the extent of methylation has been linked to cancer formation. In this study, we report a simple method to screen for the degree of DNA methylation by combined bisulfite restriction analysis (COBRA) and capillary electrophoresis with laser-induced fluorescence (CE-LIF). After treating genomic DNA with sodium bisulfite, nested-PCR amplification and endonuclease (Taq I) digestion were performed. The digested DNA fragments were then separated by capillary electrophoresis using 1.5%poly(ethylene) oxide (M_ave, 8,000,000 g/mol) in the presence of electroosmotic flow. The improvement for DNA amplification using the nested PCR described here corresponded to approximately ten cells. In addition, the level of DNA methylation shown in the electropherograms obtained corresponded to the original percentage of DNA methylation from commercial available standard sample (0-100%). The electrophoretic patterns demonstrated that the six cancer cell lines tested displayed different degrees of DNA methylation and could be differentiated by hierarchical cluster analysis. Furthermore, the DNA methylation level was eliminated after treating the cells with an anti-cancer drug (5鈥?aza-2鈥?deoxycytidine). Together, these results suggest that CE-LIF is a potentially useful and cost-effective tool for cancer diagnosis or prognosis based on the heterogeneity in a patient's DNA.