Centre for Molecular Biology and Neuroscience, Institute of Medical Microbiology, Oslo University Hospital Rikshospitalet, Oslo, Norway.
Nat Protoc. 2012 Jan 26;7(2):340-50. doi: 10.1038/nprot.2011.443.
We describe a method for the efficient and selective identification of DNA containing the 5-hydroxymethylcytosine (5-hmC) modification. This protocol takes advantage of two proteins: T4 β-glucosyltransferase (β-gt), which converts 5-hmC to β-glucosyl-5-hmC (β-glu-5-hmC), and J-binding protein 1 (JBP1), which specifically recognizes and binds to β-glu-5-hmC. We describe the steps necessary to purify JBP1 and modify this protein such that it can be fixed to magnetic beads. Thereafter, we detail how to use the JBP1 magnetic beads to obtain DNA that is enriched with 5-hmC. This method is likely to produce results similar to those of other 5-hmC pull-down assays; however, all necessary components for the completion of this protocol are readily available or can be easily and rapidly synthesized using basic molecular biology techniques. This protocol can be completed in less than 2 weeks and allows the user to isolate 5-hmC-containing genomic DNA that is suitable for analysis by quantitative PCR (qPCR), sequencing, microarray and other molecular biology assays.
我们描述了一种有效且选择性地鉴定含有 5-羟甲基胞嘧啶 (5-hmC) 修饰的 DNA 的方法。该方案利用了两种蛋白质：T4 β-葡萄糖基转移酶 (β-gt)，它将 5-hmC 转化为 β-葡萄糖基-5-hmC (β-glu-5-hmC)，以及特异性识别和结合 β-glu-5-hmC 的 J 结合蛋白 1 (JBP1)。我们描述了纯化 JBP1 和修饰该蛋白的必要步骤，以使它可以固定在磁珠上。此后，我们详细介绍了如何使用 JBP1 磁珠获得富含 5-hmC 的 DNA。该方法可能产生与其他 5-hmC 下拉测定相似的结果；然而，完成此方案所需的所有必要组件都很容易获得，或者可以使用基本的分子生物学技术轻松快速地合成。该方案可在不到 2 周的时间内完成，并允许用户分离适合定量 PCR(qPCR)、测序、微阵列和其他分子生物学测定的含有 5-hmC 的基因组 DNA。
