Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads

作者：Robertson Adam B; Dahl John Arne; Ougland Rune; Klungland Arne^*

来源：Nature Protocols, 2012, 7(2): 340-350.

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 beta-glucosyltransferase (beta-gt), which converts 5-hmC to beta-glucosyl-5-hmC (beta-glu-5-hmC), and J-binding protein 1 (JBP1), which specifically recognizes and binds to beta-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.

• 出版日期2012-2
• 单位河北医科大学