Development of a Method To Measure DNA Methylation Levels by Using Methyl CpG-Binding Protein and Luciferase-Fused Zinc Finger Protein
Development of a Method To Measure DNA Methylation Levels by Using Methyl CpG-Binding Protein and Luciferase-Fused Zinc Finger Protein
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DOI：
10.1021/ac3015774
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发表时间：
2012-10-02
影响因子：
7.4
通讯作者：
Ikebukuro, Kazunori
中科院分区：
文献类型：
--
作者：
Hiraoka, Daisuke;Yoshida, Wataru;Ikebukuro, Kazunori
DNA甲基化是转录调控的重要表观遗传事件，被认为是癌症的生物标志物。一种快速、灵敏的检测靶基因组区域DNA甲基化水平的方法可能有助于癌症的早期诊断。为了方便地检测DNA甲基化水平，我们建立了一种DNA甲基化检测系统--甲基化DNA沉淀结合DNA酶融合锌指分析（MELZA），该系统利用甲基CpG结合结构域（MBD）和DNA酶融合锌指蛋白。该系统包括以下3个步骤：（I）基于MBD的甲基化DNA沉淀，（2）靶基因组区域的PCR扩增，和（3）通过使用内切酶融合的锌指蛋白检测PCR产物量。使用该系统，我们已经准确地测量了LNCaP，PC 3，Du 145和全血细胞中雄激素受体基因启动子区域的甲基化水平。该系统不需要亚硫酸氢盐处理，并且所有步骤都可以自动化。因此，它可能是有用的，用于测量DNA甲基化水平在临床癌症诊断。
DNA methylation, which is an important epigenetic event for transcriptional regulation, is regarded as a biomarker for cancer. A rapid and sensitive method for measuring DNA methylation levels in target genomic regions may enable early diagnosis of cancer. To detect DNA methylation levels conveniently, we developed a detection system for DNA methylation, designated as methylated DNA precipitation combined luciferase-fused zinc finger assay (MELZA), which uses methyl CpG-binding domain (MBD) and luciferase-fused zinc finger protein. This system comprises the following 3 steps: (I) MBD-based methylated DNA precipitation, (2) PCR amplification of the target genomic region, and (3) detection of the PCR product quantity by using luciferase-fused zinc finger protein. Using this system, we have accurately measured methylation levels of the androgen receptor gene promoter region in LNCaP, PC3, Du145, and whole blood cells. This system does not require bisulfite treatment, and all the steps can be automated. Therefore, it might be useful for measuring DNA methylation levels in clinical cancer diagnoses.