Enzymatic approaches and bisulfite sequencing cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine in DNA

Biotechniques. 2010 Apr;48(4):317-9. doi: 10.2144/000113403.


DNA cytosine methylation (5mC) is highly abundant in mammalian cells and is associated with transcriptional repression. Recently, hydroxymethylcytosine (hmC) has been detected at high levels in certain human cell types; however, its roles are unknown. Due to the structural similarity between 5mC and hmC, it is unclear whether 5mC analyses can discriminate between these nucleotides. Here we show that 5mC and hmC are experimentally indistinguishable using established 5mC mapping methods, thereby implying that existing 5mC data sets will require careful re-evaluation in the context of the possible presence of hmC. Potential differential enrichment of 5mC and hmC DNA sequences may be facilitated using a 5mC monoclonal antibody.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5-Methylcytosine / chemistry*
  • CpG Islands
  • Cytosine / analogs & derivatives*
  • Cytosine / chemistry
  • DNA / chemistry*
  • DNA / metabolism
  • DNA Methylation
  • DNA Restriction Enzymes / metabolism
  • Genes, BRCA1
  • Humans
  • Octamer Transcription Factor-3 / genetics
  • Polymerase Chain Reaction
  • Sequence Analysis, DNA / methods*
  • Sulfites


  • Octamer Transcription Factor-3
  • POU5F1 protein, human
  • Sulfites
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine
  • Cytosine
  • DNA
  • DNA Restriction Enzymes
  • hydrogen sulfite