基于氧化石墨烯和限制性内切酶 HpaII 的荧光法检测 DNA 和 DNA 甲基化

A fluorescence method for detection of DNA and DNA methylation based on graphene oxide and restriction endonuclease HpaII.

机构信息

Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Chemistry and Chemical Engineering, Southeast University, Jiangning District, Nanjing 211189, PR China.

出版信息

Talanta. 2015 Jan;131:342-7. doi: 10.1016/j.talanta.2014.07.094. Epub 2014 Aug 6.

DOI:10.1016/j.talanta.2014.07.094

PMID:25281112

Abstract

DNA methylation plays an important role in many biological events and is associated with various diseases. Most traditional methods for detection of DNA methylation are based on the complex and expensive bisulfite method. In this paper, we report a novel fluorescence method to detect DNA and DNA methylation based on graphene oxide (GO) and restriction endonuclease HpaII. The skillfully designed probe DNA labeled with 5-carboxyfluorescein (FAM) and optimized GO concentration keep the probe/target DNA still adsorbed on the GO. After the cleavage action of HpaII the labeled FAM is released from the GO surface and its fluorescence recovers, which could be used to detect DNA in the linear range of 50 pM-50 nM with a detection limit of 43 pM. DNA methylation induced by transmethylase (Mtase) or other chemical reagents prevents HpaII from recognizing and cleaving the specific site; as a result, fluorescence cannot recover. The fluorescence recovery efficiency is closely related to the DNA methylation level, which can be used to detect DNA methylation by comparing it with the fluorescence in the presence of intact target DNA. The method for detection of DNA and DNA methylation is simple, reliable and accurate.

摘要

DNA 甲基化在许多生物事件中发挥着重要作用，与各种疾病有关。大多数传统的 DNA 甲基化检测方法都是基于复杂且昂贵的亚硫酸氢盐法。在本文中，我们报告了一种基于氧化石墨烯（GO）和限制性内切酶 HpaII 的检测 DNA 和 DNA 甲基化的新型荧光方法。经过精心设计的探针 DNA 用 5-羧基荧光素（FAM）标记，并优化了 GO 浓度，使探针/靶 DNA 仍吸附在 GO 上。在 HpaII 的切割作用下，标记的 FAM 从 GO 表面释放出来并恢复荧光，可用于检测 50 pM-50 nM 线性范围内的 DNA，检测限为 43 pM。转甲基酶（Mtase）或其他化学试剂诱导的 DNA 甲基化可阻止 HpaII 识别和切割特定位点；因此，荧光无法恢复。荧光恢复效率与 DNA 甲基化水平密切相关，可通过与完整靶 DNA 存在时的荧光进行比较来检测 DNA 甲基化。该方法用于检测 DNA 和 DNA 甲基化，简单、可靠、准确。