Investigating 5-Hydroxymethylcytosine (5hmC): The State of the Art

  • Colm E. Nestor
  • James P. Reddington
  • Mikael Benson
  • Richard R. Meehan
Part of the Methods in Molecular Biology book series (MIMB, volume 1094)


The discovery of 5-hydroxymethylcytosine (5hmC) as an abundant base in mammalian genomes has excited the field of epigenetics, and stimulated an intense period of research activity aimed at decoding its biological significance. However, initial research efforts were hampered by a lack of assays capable of specifically detecting 5hmC. Consequently, the last 3 years have seen the development of a plethora of new techniques designed to detect both global levels and locus-specific profiles of 5hmC in mammalian genomes. This research effort has culminated in the recent publication of two complementary techniques for quantitative, base-resolution mapping of 5hmC in mammalian genomes, the first true mammalian hydroxymethylomes. Here, we review the techniques currently available to researchers studying 5hmC, discuss their advantages and disadvantages, and explore the technical hurdles which remain to be overcome.

Key words

DNA methylation reprogramming 5mC 5hmC 5fC 5caC Gene regulation Epigenetics 



We thank members of the Meehan lab for advice on the development and application of 5hmC protocols. Work in RM’s lab is supported by the Medical Research Council, by the BBSRC, and by the Innovative Medicines Initiative Joint Undertaking (IMI JU) under grant agreement number 115001 (MARCAR project). URL:


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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Colm E. Nestor
    • 1
    • 2
  • James P. Reddington
    • 1
  • Mikael Benson
    • 2
  • Richard R. Meehan
    • 1
  1. 1.MRC Human Genetics Unit, IGMMUniversity of Edinburgh, Western General HospitalEdinburghUK
  2. 2.The Centre for Individualised Medication, Linkoping University HospitalLinkoping UniversityLinkopingSweden

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