title: Rapid screening of the heterogeneity of DNA methylation by single-strand conformation polymorphism and CE-LIF in the presence of electro-osmotic flow.
abstract: DNA methylation is a complex event in epigenetic studies because of both the large CpG islands present upstream of the promoter region and the different distribution of DNA methylation despite similar methylation levels. For this reason, we proposed a fast, cost-effective method for the screening of DNA methylation based on SSCP and CE-LIF. In this study, the PCR products that were amplified from bisulfite-treated genomic DNA were denatured at 94°C, followed by immediate chilling in ice water to form the ssDNA. The ssDNA were separated by 1.5% poly(ethylene oxide) (Mavg 8 000 000 Da) in the presence of EOF according to the different conformations represented by their unique methylation states. This result demonstrated that four hepatocellular carcinoma cell lines represented a different heterogeneity of DNA methylation and could be distinguished by SSCP-CE. The results obtained from SSCP-CE also corresponded with those obtained from combined bisulfide restriction analysis and methylation-sensitive high-resolution melting analysis. Therefore, the proposed SSCP-CE method may potentially be used for rapid screening for determination of the heterogeneity of DNA methylation in further epigenetic studies and clinical diagnosis.
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Meng-Hsuan
authors.initials: MH
authors.lastname: Yu
authors.forename: Ya-Chi
authors.initials: YC
authors.lastname: Huang
authors.forename: Po-Ling
authors.initials: PL
authors.lastname: Chang
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D045744:Cell Line, Tumor; D018899:CpG Islands; D004247:DNA / Q000032:analysis / Q000235:genetics; D019175:DNA Methylation; D004277:DNA, Single-Stranded / Q000032:analysis / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D004867:Equipment Design; D005453:Fluorescence; D006801:Humans; D007834:Lasers; D008113:Liver Neoplasms / Q000235:genetics*; D009995:Osmosis; D011092:Polyethylene Glycols / Q000737:chemistry; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D013447:Sulfites / Q000737:chemistry
chemical_list: D004277:DNA, Single-Stranded; D013447:Sulfites; D011092:Polyethylene Glycols; D004247:DNA; C042345:hydrogen sulfite
keywords: Cancer; Capillary electrophoresis; DNA methylation; Laser-induced fluorescence; Poly(ethylene oxide)