Methylation-sensitive high-resolution melting
Methylation-sensitive high-resolution melting
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DOI：
10.1038/nprot.2008.191
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Hansen, Lise Lotte
中科院分区：
文献类型：
--
作者：
Wojdacz, Tomasz K.;Dobrovic, Alexander;Hansen, Lise Lotte
来自亚硫酸氢钠修饰模板的 PCR 产物的基本组成依赖于甲基化。因此，甲基化和未甲基化的 PCR 产物在热变性时表现出不同的熔解曲线。甲基化敏感的高分辨率熔解 (MS-HRM) 方案基于将未知样品的 PCR 产物的熔解曲线与甲基化和未甲基化对照 DNA 衍生的 PCR 产物的熔解曲线进行比较。该方案包括使用设计用于按比例扩增甲基化和非甲基化模板的引物对亚硫酸氢盐修饰的 DNA 进行 PCR 扩增，以及随后对 PCR 产物进行高分辨率熔解分析。 MS-HRM 方案可在 3 小时内对模板 DNA 进行亚硫酸氢钠修饰后，在管内测定感兴趣位点的甲基化状态。在这里，我们提供了 MS-HRM 方案，该方案能够基于 DNA 高分辨率熔解技术进行高度灵敏、劳动力和成本效益高的单位点甲基化研究。
The base composition of PCR products derived from sodium bisulfite-modified templates is methylation dependent. Hence, methylated and unmethylated, PCR products show different melting profiles when subjected to thermal denaturation. The methylation-sensitive high-resolution melting (MS-HRM) protocol is based on the comparison of the melting profiles of PCR products from unknown samples with profiles specific for PCR products derived from methylated and unmethylated control DNAs. The protocol consists of PCR amplification of bisulfite-modified DNA with primers designed to proportionally amplify both methylated and unmethylated templates and subsequent high-resolution melting analysis of the PCR product. The MS-HRM protocol allows in-tube determination of the methylation status of the locus of interest following sodium bisulfite modification of template DNA in less than 3 h. Here, we provide a protocol for MS-HRM, which enables highly sensitive, labor- and cost-efficient single-locus methylation studies on the basis of DNA high-resolution melting technology.