Electrochemical DNA Methylation Detection for Enzymatically Digested CpG Oligonucleotides
Electrochemical DNA Methylation Detection for Enzymatically Digested CpG Oligonucleotides
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DOI：
10.1021/ac201761c
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发表时间：
2011-10-15
影响因子：
7.4
通讯作者：
Niwa, Osamu
中科院分区：
文献类型：
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作者：
Kato, Dai;Goto, Keisuke;Niwa, Osamu
我们描述了DNA甲基化的电化学检测，通过直接氧化的5-甲基胞嘧啶（mC）和胞嘧啶（C）在5 '-CG-3'序列（CpG）寡核苷酸后，使用溅射纳米碳膜电极酶切较长的CpG寡核苷酸与核酸内切酶P1。较长的CpG寡核苷酸的直接电化学不足以获得这些碱基的氧化电流，因为富含CG的序列抑制了较长的CpG寡核苷酸中每个碱基的直接氧化，这是由于构象结构及其非常低的扩散系数。为了在相对较长的CpG寡核苷酸中以更好的定量和灵敏度检测C甲基化，我们成功地使用内切核酸酶P1消化靶CpG寡核苷酸并产生相同的单核苷酸2 '-脱氧核糖核苷5'-单磷酸（5 '-dNMP）。与未经P1处理的结果相比，我们获得了4.4倍的灵敏度和更宽的浓度范围，用于mC检测，分辨率能够检测CpG寡核苷酸（60聚体）中微小的甲基化胞嘧啶差异。
We describe the electrochemical detection of DNA methylation through the direct oxidation of both 5-methylcytosine (mC) and cytosine (C) in 5'-CG-3' sequence (CpG) oligonudeotides 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 mononucleotide2'-deoxyribonucleoside 5'-monophosphate (5'-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 oligonudeotides (60mer).