Methods of 5-hmC Detection and Analysis

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Since restriction enzyme based protocols are effective for mapping 5-mC, a method has been developed to differentiate 5-mC from 5-hmC utilizing a glucosylation step that only modifies the 5-hmC followed by a restriction enzyme digestion. This covalent attachment of a glucose results in differential substrate specificity for 5-mC versus 5-hmC. When 5-hmC occurs at the internal CG of CCGG, this glucosylation of internal CG blocks the recognition site for the methylation insensitive restriction enzyme MspI. PCR analysis following enzymatic digestion reveals a product indicating presence of 5-hmC. Quantitative PCR based methods can be used to determine relative abundance of C, 5-mC and 5-hmC at the internal CpG site of CCGG sequence.

The MspJI family of restriction enzymes is dually-dependent on methylation (5-mC) or hydroxymethylation ( 5-hmC) for cleavage. These enzymes excise 32-base pair DNA fragments containing a centrally located 5-mC or 5-hmC residue that can be subsequently purified and sequenced. Due to the known position of this epigenetic modification, bisulfite conversion is not required prior to downstream analysis (1).

The PvuRts1I family of restriction enzymes can be used for near single residue resolution mapping of genomic 5-hmC. A PvuRts1I family member known as AbaSI, shows ~10,000 fold higher specificity between glucosylated 5-hmC and 5-mC and could possibly be used for base resolution mapping of 5-hmC in the genome (2).

There are also two affinity based enrichment methods to capture and analyze 5-hmC. hMeDIP utilizes 5-hMC specific antibodies (3,4). Similarly, J-binding protein pull down assay can enrich 5-hmC DNA from genomic DNA (5). In this protocol, β -glucosyltransferase treated 5-hmC is selectively precipitated by J-binding protein coupled to magnetic beads. J-binding protein is derived from the organism Crithidia fasciculate, and has affinity for β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC) containing DNA. Both enrichment methods can be followed downstream by qPCR, microarray or sequencing.


  1. Cohen-Karni D et al (2011) Proc Natl Acad Sci USA108(27):11040-5. PMID: 21690366
  2. Wang, H. et al. (2011) Nucleic Acids Res. 39(21):9294-305. PMID: 21813453
  3. Haffner MC (2011) Oncotarget (8):627-37. PMID: 21896958
  4. Stroud H. (2011) Genome Biol. 12(6):R54. PMID: 21689397
  5. Robertson AB (2012) Nat Protoc. 7(2):340-50. PMID: 22281869