Department of Medicine and Science of Aging, University G. D'Annunzio, Chieti, Italy.
Electrophoresis. 2013 Aug;34(15):2275-80. doi: 10.1002/elps.201300091. Epub 2013 Jul 9.
Analytical methods for quantification of 5'-methylcytosine in genomes are important tools to investigate epigenetic changes in gene expression during development, differentiation, aging, or cancer. Here, we report a novel genomic methylation content assay based on enzymatic hydrolysis of DNA and MEKC separation of 5'-deoxyribonucleoside monophosphates (dNMP) using the cationic surfactant CTAB as pseudostationary phase. Calf Thymus DNA was used during method development to determine electrophoretic parameters and electrolyte composition for a complete separation between 2'-deoxycytosine-5'-monophosphate and 2'-deoxy-5'-methylcytosine 5'-monophosphate (d5mCMP). Methylated and not methylated oligonucleotides were used to confirm the identity of each peak and evaluate analytical parameters of the method. The LOD of the method was found to be 12.5 pmol/μL for d5mCMP.
用于定量基因组中 5'-甲基胞嘧啶的分析方法是研究发育、分化、衰老或癌症过程中基因表达的表观遗传变化的重要工具。在这里，我们报告了一种基于 DNA 酶水解和 MEKC 分离 5'-脱氧核苷一磷酸（dNMP）的新型基因组甲基化含量测定方法，使用阳离子表面活性剂 CTAB 作为伪固定相。在方法开发过程中使用小牛胸腺 DNA 来确定电泳参数和电解质组成，以实现 2'-脱氧胞嘧啶-5'-单磷酸和 2'-脱氧-5'-甲基胞嘧啶 5'-单磷酸（d5mCMP）之间的完全分离。使用甲基化和非甲基化的寡核苷酸来确认每个峰的身份，并评估该方法的分析参数。该方法的检出限（LOD）为 12.5 pmol/μL 的 d5mCMP。
