title: Global DNA Methylation Detection System Using MBD-Fused Luciferase Based on Bioluminescence Resonance Energy Transfer Assay.
abstract: DNA methylation plays an important role in the regulation of gene expression. In normal cells, transposable elements that constitute approximately 45% of the human genome are highly methylated to silence their expression. In cancer cells, transposable elements are hypomethylated; therefore, global DNA methylation level is considered as a biomarker for cancer diagnostics. In this study, a homogeneous assay for measuring global DNA methylation level based on bioluminescence resonance energy transfer (BRET) was developed using methyl-CpG binding domain (MBD)-fused luciferase. In this assay, the MBD-luciferase recognizes methylated CpG, thus, BRET between the luciferase and fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the DNA methylation level of the target DNA. Moreover, the BRET signal was correlated with the LINE1 DNA methylation level on human genomic DNA, as determined by the bisulfite method. These results indicate that the global DNA methylation level of human genomic DNA could be detected simply by measuring the BRET signal.
authors.affiliation: School of Bioscience and Biotechnology, Tokyo University of Technology , 1404-1 Katakuramachi, Hachioji, Tokyo 192-0982, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Yoshida
authors.affiliation: School of Bioscience and Biotechnology, Tokyo University of Technology , 1404-1 Katakuramachi, Hachioji, Tokyo 192-0982, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Baba
authors.affiliation: School of Bioscience and Biotechnology, Tokyo University of Technology , 1404-1 Katakuramachi, Hachioji, Tokyo 192-0982, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Karube
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004735:Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008958:Models, Molecular; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics; D000072417:Protein Domains; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases