基于甲基化核酸内切酶的甲基转移酶活性的电化学检测研究  

Electrochemical detection of methyltransferase activity based on methylation endonuclease

作　　者：白雪娇 鲁理平[1] 康天放[1] BAI Xue-jiao;LU Li-ping;KANG Tian-fang(College of Environmental and Energy Engineering,Beijing University of Technology,Beijing 100124,China)

机构地区：[1]北京工业大学环境与能源工程学院

出　　处：《化学研究与应用》2019年第6期1079-1085,共7页Chemical Research and Application

基　　金：国家自然科学基金项目(21876005)资助

摘　　要：由DNA甲基转移酶(DNMT1)催化的DNA甲基化在许多生物过程中起重要作用,包括基因转录,基因组印记和细胞分化[1]。本文利用特异性酶切法成功开发了一种有效的测定DNMT1的电化学方法。首先,将硫醇修饰的双链DNA(dsDNA)自组装固定在金电极上,随后DNMT1识别dsDNA半甲基化序列并实现完全甲基化,最后通过甲基化特异性限制酶(BSSHⅡ)剪切,未发生完全甲基化的碱基序列将被剪切并移除电极表面。利用差分脉冲伏安法(DPV)分别测定剪切前后的亚甲基蓝(MB)还原电流。在最佳优化条件下,该方法线性范围为0.5 nM^50 nM,检测限为0.5 nM。这种方法可以简单有效地检测DNA甲基化和DNMT1活性,在DNA甲基化快速检测和基因毒理分析方面具有一定的实际意义。DNA methylation catalyzed by DNA methyltransferase(DNMT1)plays an important role in many biological processes,including gene transcription,genomic imprinting,and cellular differentiation [1].In this paper,an efficient electrochemical method for the determination of DNA methylation and DNMT1 was successfully developed by restriction endonuclease method.First,the thiol-modified double-stranded DNA(dsDNA)was self-assembled on gold electrode.Through the activity of DNMT1,the hemimethylated CG recognition sequence of the dsDNA are methylated and unmethylated sequence was cleaved and removed after the subsequently treatment with methylation-specific restriction enzyme(BSSH Ⅱ).We measured the methylene blue(MB)reduction current before and after cleaved by differential pulse voltammetry(DPV)respectively.Under optimal conditions,the method shows a linear range of 0.5 nM^50 nM with a detection limit of 0.5 nM.Since this method can detect DNA methylation and DNMT1 activity simply and effectively,it is practical in DNA methylation rapid detection and genotoxic analysis.

关 键 词：DNA甲基化 甲基转移酶活性 特异性酶切法 亚甲基蓝 

分 类 号：O657.1[理学—分析化学]