title: Determination of DNA methylation using electrochemiluminescence with surface accumulable coreactant.
abstract: Cytosine methylation in DNA was determined by an enzyme linked immunosorbent assay (ELISA) with electrochemiluminescence (ECL) detection and employed for the DNA methylation assay of a long and real genomic sample for the first time. The developed method employed an antimethyl cytosine antibody labeled with acetylcholinesterase, which was added to recognize single methylated cytosine in a DNA oligomer. The acetylcholinesterase converted acetylthiocholine (substrate) to thiocholine (product), which was accumulated on a gold electrode surface via gold-thiol binding. This surface accumulated preconcentration made it possible to observe bright and distinctive ECL by applying a potential to the gold electrode in the presence of a tris(2,2-bipyridyl)ruthenium complex luminophore when the analyte DNA contained a methylation region. Methyl-cytosine was measured quantitatively in the 1-100 pmol range, which exhibits sufficiently high sensitivity to achieve real DNA measurements without amplification by a polymerase chain reaction (PCR). The proposed ECL method also exhibited high selectivity for methyl-cytosine against nonmethylated cytosine, guanine, thymine, and adenine nucleotides. Finally, original and methylated DNA samples were clearly distinguished with our method using a real DNA bacteriophage sample (48,502 base pairs).
authors.affiliation: National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566. r.kurita@aist.go.jp
authors.forename: Ryoji
authors.initials: R
authors.lastname: Kurita
authors.forename: Kumi
authors.initials: K
authors.lastname: Arai
authors.forename: Kohei
authors.initials: K
authors.lastname: Nakamoto
authors.forename: Dai
authors.initials: D
authors.lastname: Kato
authors.forename: Osamu
authors.initials: O
authors.lastname: Niwa
mesh_terms: D015082:2,2'-Dipyridyl / Q000737:chemistry; D001435:Bacteriophages / Q000235:genetics; D003596:Cytosine / Q000737:chemistry*; D019175:DNA Methylation; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D004563:Electrochemistry; D008163:Luminescent Measurements; D016133:Polymerase Chain Reaction; D012428:Ruthenium / Q000737:chemistry
chemical_list: D004279:DNA, Viral; D015082:2,2'-Dipyridyl; D012428:Ruthenium; D003596:Cytosine