We report two restriction enzyme-based approaches for generating clean locus-specific unmethylated controls for methylation-sensitive high-resolution melting (MS-HRM) analyses. These unmethylated standards are derived from DNA treated with the demethylating agent 5-aza-2-deoxycytidine (5-Aza-dc). By using them, we overcome a limitation of 5-Aza-dc treatment - incomplete demethylation at various genomic regions. When 5-Aza-dc-treated DNA is used directly as unmethylated MS-HRM standard, partially demethylated DNA can give false methylation results. MS-HRM assay differentiates between methylated and unmethylated bisulfite-treated DNA based on the different melting profiles of PCR products amplified from them. To estimate test sample methylation levels, test sample melting profiles are compared to those of methylation standards. With our pure unmethylated controls, adequate standards of known methylation levels can be prepared for single-locus MS-HRM.

中文翻译：

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改进的位点特异性未甲基化的MS-HRM分析对照，衍生自5-氮杂-2-脱氧胞苷处理的DNA。

我们报告了两种基于限制酶的方法，用于生成甲基化敏感的高分辨率熔解（MS-HRM）分析的干净的基因座特异性未甲基化的控件。这些未甲基化的标准品衍生自用脱甲基剂5-氮杂-2-脱氧胞苷（5-氮杂-dc）处理的DNA。通过使用它们，我们克服了5-Aza-dc处理的局限性-在各个基因组区域不完全脱甲基。当将5-Aza-dc处理的DNA直接用作未甲基化的MS-HRM标准品时，部分去甲基化的DNA会产生错误的甲基化结果。MS-HRM分析根据扩增后的PCR产物的不同熔解曲线，区分甲基化和未甲基化的亚硫酸氢盐处理的DNA。为了估计测试样品的甲基化水平，将测试样品的熔解曲线与甲基化标准品的熔解曲线进行比较。