Document Detail


The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine.
MedLine Citation:
PMID:  23018193     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
5-Hydroxymethylcytosine (5hmC) is a recently discovered base in the mammalian genome, produced upon oxidation of 5-methylcytosine (5mC) in a process catalyzed by TET proteins. The biological functions of 5hmC and further oxidation products of 5mC are under intense investigation, as they are likely intermediates in DNA demethylation pathways. Here we describe a novel protocol to profile 5hmC at a genome-wide scale. This approach is based on sodium bisulfite-mediated conversion of 5hmC to cytosine-5-methylenesulfonate (CMS); CMS-containing DNA fragments are then immunoprecipitated using a CMS-specific antiserum. The anti-CMS technique is highly specific with a low background, and is much less dependent on 5hmC density than anti-5hmC immunoprecipitation (IP). Moreover, it does not enrich for CA and CT repeats, as noted for 5hmC DNA IP using antibodies to 5hmC. The anti-CMS protocol takes 3 d to complete.
Authors:
Yun Huang; William A Pastor; Jorge A Zepeda-Martínez; Anjana Rao
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-09-27
Journal Detail:
Title:  Nature protocols     Volume:  7     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-09-28     Completed Date:  2013-01-28     Revised Date:  2013-10-17    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  1897-908     Citation Subset:  IM    
Affiliation:
La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.
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MeSH Terms
Descriptor/Qualifier:
Antibodies
Base Sequence
Cytosine / analogs & derivatives*,  analysis,  chemistry
DNA Primers
Genomics / methods*
Immunoprecipitation / methods
Molecular Sequence Data
Sulfites / chemistry
Grant Support
ID/Acronym/Agency:
AI44432/AI/NIAID NIH HHS; HD065812/HD/NICHD NIH HHS; R01 AI044432/AI/NIAID NIH HHS; R01 CA151535/CA/NCI NIH HHS; R01 HD065812/HD/NICHD NIH HHS
Chemical
Reg. No./Substance:
0/Antibodies; 0/DNA Primers; 0/Sulfites; 1123-95-1/5-hydroxymethylcytosine; 71-30-7/Cytosine; 7631-90-5/sodium bisulfite
Comments/Corrections

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