Electrochemical DNA Methylation Detection for Enzymatically Digested CpG Oligonucleotides

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• 作者：Dai Kato ; Keisuke Goto ; Shin-ichiro Fujii ; Akiko Takatsu ; Shigeru Hirono ; Osamu Niwa
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：October 15, 2011
• 年：2011
• 卷：83
• 期：20
• 页码：7595-7599
• 全文大小：801K
• 年卷期：v.83,no.20(October 15, 2011)
• ISSN：1520-6882

文摘

We describe the electrochemical detection of DNA methylation through the direct oxidation of both 5-methylcytosine (mC) and cytosine (C) in 5鈥?CG-3鈥?sequence (CpG) oligonucleotides using a sputtered nanocarbon film electrode after digesting a longer CpG oligonucleotide with endonuclease P1. Direct electrochemistry of the longer CpG oligonucleotides was insufficient for obtaining the oxidation currents of these bases because the CG rich sequence inhibited the direct oxidation of each base in the longer CpG oligonucleotides, owing to the conformational structure and its very low diffusion coefficient. To detect C methylation with better quantitativity and sensitivity in the relatively long CpG oligonucleotides, we successfully used an endonuclease P1 to digest the target CpG oligonucleotide and yield an identical mononucleotide 2鈥?deoxyribonucleoside 5鈥?monophosphate (5鈥?dNMP). Compared with results obtained without P1 treatment, we achieved 4.4 times higher sensitivity and a wider concentration range for mC detection with a resolution capable of detecting a subtle methylated cytosine difference in the CpG oligonucleotides (60mer).