Site-specific detection of DNA methylation utilizing mCpG-SEER

Cliff I. Stains, Jennifer L. Furman, David J. Segal, Indraneel Ghosh

Research output: Contribution to journalArticlepeer-review

68 Scopus citations

Abstract

Currently there are no direct methods for the sequence-specific detection of DNA-methylation at CpG dinucleotides, which provide a possible diagnostic marker for cancer. Toward this goal, we present a methodology termed mCpG-SEquence Enabled Reassembly (mCpG-SEER) of proteins utilizing a split green fluorescent protein (GFP) tethered to specific DNA recognition elements. Our system, mCpG-SEER, employs a zinc-finger attached to one-half of GFP to target a specific sequence of dsDNA, while a methyl-CpG binding domain protein attached to the complementary half of GFP targets an adjacent methylated CpG dinucleotide site. We demonstrate that the presence of both DNA sites is necessary for the reassembly and concomitant fluorescence of the reassembled GFP. We further show that the GFP-dependent fluorescence reaches a maximum when the methyl-CpG and zinc-finger sites are separated by two base pairs and the fluorescence signal is linear to 5 pmol of methylated target DNA. Finally, the specificity of this reporter system, mCpG-SEER, was found to be >40-fold between a methylated versus a nonmethylated CpG target site.

Original languageEnglish (US)
Pages (from-to)9761-9765
Number of pages5
JournalJournal of the American Chemical Society
Volume128
Issue number30
DOIs
StatePublished - Aug 2 2006

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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