Epigenetics ISP, The Babraham Institute, Cambridge, UK.
MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
Methods Mol Biol. 2021;2272:45-63. doi: 10.1007/978-1-0716-1294-1_4.
5-Hydroxymethylcytosine (5hmC) is an abundant DNA modification in human and mouse brain, as well as in embryonic stem cells, while severely depleted in multiple types of cancer. Assays for 5hmC detection and quantification, both on a locus-specific and global level, are limited in number and often resource-intensive. Immunodetection of 5hmC through antibodies remains a cost-effective and widely accessible approach. This chapter describes an ELISA-based protocol for 5hmC detection and quantification in genomic or in vitro modified DNA. It is based on the passive adsorption of DNA onto a solid polystyrene surface and the specific detection of 5hmC, which generates a measurable chemiluminescent signal, proportional to the amount of immobilized 5hmC. The assay utilizes a standard curve for interpolation of 5hmC percentage and a loading standard for monitoring loading precision.
5-羟甲基胞嘧啶（5hmC）是人类和小鼠大脑以及胚胎干细胞中丰富的 DNA 修饰物，而在多种类型的癌症中严重缺失。5hmC 的检测和定量分析方法，无论是在特定基因座还是在全基因组水平上，数量都很有限，而且通常资源密集。通过抗体进行 5hmC 的免疫检测仍然是一种具有成本效益且广泛适用的方法。本章描述了一种基于 ELISA 的方法，用于检测和定量基因组或体外修饰的 DNA 中的 5hmC。它基于 DNA 被动吸附到聚苯乙烯固体表面上，以及 5hmC 的特异性检测，这会产生可测量的化学发光信号，与固定化 5hmC 的量成正比。该测定法利用标准曲线进行 5hmC 百分比的内插，并利用加载标准来监测加载精度。
