Development of an electrochemical detection system for measuring DNA methylation levels using methyl CpG-binding protein and glucose dehydrogenase-fused zinc finger protein
Development of an electrochemical detection system for measuring DNA methylation levels using methyl CpG-binding protein and glucose dehydrogenase-fused zinc finger protein
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DOI：
10.1016/j.bios.2016.09.060
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发表时间：
2017-07-15
影响因子：
12.6
通讯作者：
Ikebukuro, Kazunori
中科院分区：
文献类型：
--
作者：
Lee, Jinhee;Yoshida, Wataru;Ikebukuro, Kazunori
特定基因区域的DNA甲基化水平被认为是一种新型的诊断生物标志物，诊断需要小型化、快速的检测系统。在这里，我们开发了一种简单的 DNA 甲基化电化学检测系统，使用甲基 CpG 结合域 (MBD) 和葡萄糖脱氢酶 (GDH) 融合锌指蛋白。该分析系统由三个步骤组成：(1) 通过 MBD 收集甲基化 DNA，(2) 对收集的甲基化 DNA 中的目标基因组区域进行 PCR 扩增，以及 (3) 使用 GDH 融合锌指蛋白对 PCR 产物进行电化学检测。利用该系统，我们成功测量了从 PC3 和 TSU-PR1 癌细胞系中提取的 106 个基因组 DNA 拷贝中雄激素受体基因启动子区域的甲基化水平。由于该检测系统不需要序列分析或酶消化，因此可以通过简单的程序在 3 小时内测量 DNA 甲基化水平。
DNA methylation level at a certain gene region is considered as a new type of biomarker for diagnosis and its miniaturized and rapid detection system is required for diagnosis. Here we have developed a simple electrochemical detection system for DNA methylation using methyl CpG-binding domain (MBD) and a glucose dehydrogenase (GDH)-fused zinc finger protein. This analytical system consists of three steps: (1) methylated DNA collection by MBD, (2) PCR amplification of a target genomic region among collected methylated DNA, and (3) electrochemical detection of the PCR products using a GDH-fused zinc finger protein. With this system, we have successfully measured the methylation levels at the promoter region of the androgen receptor gene in 106 copies of genomic DNA extracted from PC3 and TSU-PR1 cancer cell lines. Since no sequence analysis or enzymatic digestion is required for this detection system, DNA methylation levels can be measured within 3 h with a simple procedure.