Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 128, Issue 30, Pages 9761-9765Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ja060681j
Keywords
-
Categories
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available