University of Maribor, Faculty of Chemistry & Chemical Engineering, Smetanova ulica 17, 2000 Maribor, Slovenia.
University of Maribor, Faculty of Medicine, Center for Human Molecular Genetics & Pharmacogenomics, Taborska ulica 8, 2000 Maribor, Slovenia.
Biotechniques. 2019 Mar;66(3):150-153. doi: 10.2144/btn-2018-0161. Epub 2019 Feb 14.
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.
我们报告了两种基于限制性内切酶的方法，用于生成用于甲基化敏感高分辨率熔解（MS-HRM）分析的清洁、位点特异性非甲基化对照。这些非甲基化标准品源自用去甲基化剂 5-氮杂-2-脱氧胞苷（5-Aza-dc）处理的 DNA。通过使用这些标准品，我们克服了 5-Aza-dc 处理的一个局限性 - 在各种基因组区域的不完全去甲基化。当直接将 5-Aza-dc 处理的 DNA 用作非甲基化 MS-HRM 标准时，部分去甲基化的 DNA 可能会给出错误的甲基化结果。MS-HRM 分析基于从它们扩增的 PCR 产物的不同熔解曲线来区分甲基化和非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。使用我们的纯非甲基化对照，可以为单基因座 MS-HRM 制备具有已知甲基化水平的充足标准品。
