title: Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads.
abstract: 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.
authors.affiliation: Centre for Molecular Biology and Neuroscience, Institute of Medical Microbiology, Oslo University Hospital Rikshospitalet, Oslo, Norway.
authors.forename: Adam B
authors.initials: AB
authors.lastname: Robertson
authors.forename: John Arne
authors.initials: JA
authors.lastname: Dahl
authors.forename: Rune
authors.initials: R
authors.lastname: Ougland
authors.forename: Arne
authors.initials: A
authors.lastname: Klungland
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification; D005964:Glucosyltransferases / Q000737:chemistry; D060328:Magnetic Phenomena; D008863:Microspheres
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D005964:Glucosyltransferases