Development of an electrochemical detection system for measuring DNA methylation levels using methyl CpG-binding protein and glucose dehydrogenase-fused zinc finger protein.
DNA methylation level at a certain gene region is considered as a new type of biomarker for diagnosis and its miniaturized and rapid detection system is required for diagnosis. Here we have developed a simple electrochemical detection system for DNA methylation using methyl CpG-binding domain (MBD) and a glucose dehydrogenase (GDH)-fused zinc finger protein. This analytical system consists of three steps: (1) methylated DNA collection by MBD, (2) PCR amplification of a target genomic region among collected methylated DNA, and (3) electrochemical detection of the PCR products using a GDH-fused zinc finger protein. With this system, we have successfully measured the methylation levels at the promoter region of the androgen receptor gene in 106copies of genomic DNA extracted from PC3 and TSU-PR1 cancer cell lines. Since no sequence analysis or enzymatic digestion is required for this detection system, DNA methylation levels can be measured within 3h with a simple procedure.
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-Cho, Koganei, Tokyo 184-8588, Japan.
Jinhee
J
Lee
School of Biotechnology & Bioscience, Tokyo University of Technology, 1404-1 Katakura-Machi, Hachioji, Tokyo 192-0982, Japan.
Wataru
W
Yoshida
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-Cho, Koganei, Tokyo 184-8588, Japan.
Koichi
K
Abe
Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, 2-10-1 Okura-Cho, Setagaya-Ku, Tokyo 157-8535, Japan.
Kazuhiko
K
Nakabayashi
Department of Urology, University of Miyazaki, 5200, Kihara, Kiyotake-Cho, Miyazaki 889-1692, Japan.
Hironobu
H
Wakeda
Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, 2-10-1 Okura-Cho, Setagaya-Ku, Tokyo 157-8535, Japan.
Kenichiro
K
Hata
Univ Lyon, Université Lyon1, CNRS, INSA, CPE-Lyon, ICBMS, UMR 5246, 43 Bd Du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
Christophe A
CA
Marquette
Univ Lyon, Université Lyon1, CNRS, INSA, CPE-Lyon, ICBMS, UMR 5246, 43 Bd Du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
Loïc J
LJ
Blum
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-Cho, Koganei, Tokyo 184-8588, Japan.
Koji
K
Sode
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-Cho, Koganei, Tokyo 184-8588, Japan. Electronic address: ikebu@cc.tuat.ac.jp.
Kazunori
K
Ikebukuro
D015374:Biosensing Techniques; D018899:CpG Islands / Q000235:genetics; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D011401:Promoter Regions, Genetic
D004268:DNA-Binding Proteins; D004247:DNA; D042843:Glucose 1-Dehydrogenase
DNA methylation; Diagnosis; Electrochemical detection; Glucose dehydrogenase; Methyl-CpG binding domain; Zinc finger protein