title: Development of a method to measure DNA methylation levels by using methyl CpG-binding protein and luciferase-fused zinc finger protein.
abstract: DNA methylation, which is an important epigenetic event for transcriptional regulation, is regarded as a biomarker for cancer. A rapid and sensitive method for measuring DNA methylation levels in target genomic regions may enable early diagnosis of cancer. To detect DNA methylation levels conveniently, we developed a detection system for DNA methylation, designated as methylated DNA precipitation combined luciferase-fused zinc finger assay (MELZA), which uses methyl CpG-binding domain (MBD) and luciferase-fused zinc finger protein. This system comprises the following 3 steps: (1) MBD-based methylated DNA precipitation, (2) PCR amplification of the target genomic region, and (3) detection of the PCR product quantity by using luciferase-fused zinc finger protein. Using this system, we have accurately measured methylation levels of the androgen receptor gene promoter region in LNCaP, PC3, Du145, and whole blood cells. This system does not require bisulfite treatment, and all the steps can be automated. Therefore, it might be useful for measuring DNA methylation levels in clinical cancer diagnoses.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Hiraoka
authors.forename: Wataru
authors.initials: W
authors.lastname: Yoshida
authors.forename: Koichi
authors.initials: K
authors.lastname: Abe
authors.forename: Hironobu
authors.initials: H
authors.lastname: Wakeda
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Hata
authors.forename: Kazunori
authors.initials: K
authors.lastname: Ikebukuro
mesh_terms: D004247:DNA / Q000032:analysis* / Q000235:genetics; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D016133:Polymerase Chain Reaction; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; C066141:MBD1 protein, human; D014157:Transcription Factors; D004247:DNA; D008156:Luciferases; D005982:Glutathione Transferase; D005978:Glutathione