Site-specific detection of DNA methylation utilizing mCpG-SEER.
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.
Stains
Cliff I
CI
Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.
Furman
Jennifer L
JL
Segal
David J
DJ
Ghosh
Indraneel
I
D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D018899:CpG Islands; D004247:DNA / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012680:Sensitivity and Specificity
D004268:DNA-Binding Proteins; C489325:MBD2 protein, human; D004247:DNA