Site-Specific Detection of DNA Methylation Utilizing mCpG-SEER

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• 作者：Cliff I. Stains ; Jennifer L. Furman ; David J. Segal ; Indraneel Ghosh
• 刊名：Journal of the American Chemical Society
• 出版年：2006
• 出版时间：August 2, 2006
• 年：2006
• 卷：128
• 期：30
• 页码：9761 - 9765
• 全文大小：302K
• 年卷期：v.128,no.30(August 2, 2006)
• ISSN：1520-5126

文摘

Currently there are no direct methods for the sequence-specific detection of DNA-methylationat CpG dinucleotides, which provide a possible diagnostic marker for cancer. Toward this goal, we presenta methodology termed mCpG-SEquence Enabled Reassembly (mCpG-SEER) of proteins utilizing a splitgreen 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 methylatedCpG dinucleotide site. We demonstrate that the presence of both DNA sites is necessary for the reassemblyand concomitant fluorescence of the reassembled GFP. We further show that the GFP-dependentfluorescence reaches a maximum when the methyl-CpG and zinc-finger sites are separated by two basepairs and the fluorescence signal is linear to 5 pmol of methylated target DNA. Finally, the specificity ofthis reporter system, mCpG-SEER, was found to be >40-fold between a methylated versus a nonmethylatedCpG target site.