title: The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine.
abstract: 5-Hydroxymethylcytosine (5hmC) is a newly discovered DNA base present at detectable levels in most mammalian cell types and tissues. It is generated by Tet-enzyme-mediated oxidation of 5-methylcytosine (5mC). 5hmC is important both because of its potential role in regulating gene expression and because it may be an intermediate in DNA demethylation. Here we describe a technique termed GLIB (glucosylation, periodate oxidation and biotinylation), which combines several enzymatic and chemical modification steps to attach biotin to 5hmC. Biotin-containing genomic DNA fragments are then enriched using streptavidin beads, eluted and sequenced. GLIB is capable of quantitatively tagging and precipitating fragments containing a single 5hmC molecule. Sample preparation and GLIB can be conducted in 2-3 d.
authors.affiliation: La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Pastor
authors.forename: Yun
authors.initials: Y
authors.lastname: Huang
authors.forename: Hope R
authors.initials: HR
authors.lastname: Henderson
authors.forename: Suneet
authors.initials: S
authors.lastname: Agarwal
authors.forename: Anjana
authors.initials: A
authors.lastname: Rao
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D001483:Base Sequence; D019921:Biotinylation / Q000379:methods; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D023281:Genomics / Q000379:methods*; D005947:Glucose / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010504:Periodic Acid / Q000737:chemistry
chemical_list: D010504:Periodic Acid; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; C009288:metaperiodate; D005947:Glucose