Determination of DNA methylation using electrochemiluminescence with surface accumulable coreactant

Anal Chem. 2012 Feb 21;84(4):1799-803. doi: 10.1021/ac202692f. Epub 2012 Feb 2.

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 2,2'-Dipyridyl / chemistry
  • Bacteriophages / genetics
  • Cytosine / chemistry*
  • DNA Methylation*
  • DNA, Viral / analysis*
  • DNA, Viral / genetics
  • Electrochemistry*
  • Luminescent Measurements*
  • Polymerase Chain Reaction
  • Ruthenium / chemistry

Substances

  • DNA, Viral
  • 2,2'-Dipyridyl
  • Ruthenium
  • Cytosine