Single-molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong, China.
Anal Chem. 2012 Jul 17;84(14):6199-205. doi: 10.1021/ac301186j. Epub 2012 Jul 2.
Sensitive and specific detection of DNA methylation in CpG sites of genomic DNA is imperative for rapid epigenetic evaluation and early cancer diagnosis. Here, we employ for the first time the thermostable ligation for methylated DNA discrimination and hyperbranched rolling circle amplification (HRCA) for signal enhancement, without the need for restriction enzymes, PCR amplification, or fluorescence-labeled probes. After bisulfite treatment of methylated DNA, the methylation-specific linear padlock probe can be circularized only in the presence of methylated DNA and serves subsequently as a template for HRCA, whose products are easily detected using SYBR Green I and a standard fluorometer. While in the presence of unmethylated DNA, the linear padlock probe cannot be circularized because of the defectively matched substrate, and no HRCA occurs. This ligation-mediated HRCA-based method exhibits excellent specificity and high sensitivity with a detection limit of 0.8 fM and a detection range of 4 orders of magnitude, and it can even distinguish as low as 0.01% methylation level from the mixture, which is superior to most currently used methods for DNA methylation assay. This method can be further applied to analyze genomic DNA in human lung cancer cells.
对基因组 DNA 中 CpG 位点的 DNA 甲基化进行敏感和特异性检测，对于快速进行表观遗传学评估和早期癌症诊断至关重要。在这里，我们首次采用热稳定的连接用于甲基化 DNA 区分和超支化滚环扩增（HRCA）进行信号增强，而无需限制酶、PCR 扩增或荧光标记探针。在甲基化 DNA 的亚硫酸氢盐处理后，只有在存在甲基化 DNA 的情况下，甲基化特异性线性发夹探针才能被环化，并随后作为 HRCA 的模板，其产物可以使用 SYBR Green I 和标准荧光计轻松检测。而在存在非甲基化 DNA 的情况下，由于底物匹配缺陷，线性发夹探针不能被环化，也不会发生 HRCA。这种基于连接介导的 HRCA 的方法具有优异的特异性和高灵敏度，检测限为 0.8 fM，检测范围为 4 个数量级，甚至可以从混合物中区分低至 0.01%的甲基化水平，优于大多数目前用于 DNA 甲基化分析的方法。该方法可进一步应用于分析人类肺癌细胞中的基因组 DNA。
