Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Anal Chem. 2011 Oct 15;83(20):7595-9. doi: 10.1021/ac201761c. Epub 2011 Sep 16.
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) 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'-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 oligonucleotides (60mer).
我们描述了一种电化学检测方法，可通过使用溅射纳米碳膜电极直接氧化 5'-CG-3' 序列（CpG）寡核苷酸中的 5-甲基胞嘧啶（mC）和胞嘧啶（C），从而检测 DNA 甲基化。在使用内切酶 P1 消化较长的 CpG 寡核苷酸后，CpG 寡核苷酸中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 的序列会由于构象结构及其非常低的扩散系数而抑制较长的 CpG 寡核苷酸中每个碱基的直接氧化。为了在相对较长的 CpG 寡核苷酸中获得更好的定量和灵敏度检测 C 甲基化，我们成功地使用内切酶 P1 消化靶 CpG 寡核苷酸，并生成相同的单核苷酸 2'-脱氧核糖核苷酸 5'-单磷酸（5'-dNMP）。与未经 P1 处理的结果相比，我们实现了 4.4 倍的更高灵敏度和更宽的 mC 检测浓度范围，分辨率能够检测 CpG 寡核苷酸中微妙的甲基化胞嘧啶差异（60mer）。
