5-hmC Detection & Analysis

In mammalian genomes, 5-mC can be enzymatically oxidized to 5-hmC (5-hydroxymethylcytosine). There is intense scientific curiosity on identifying the epigenetic mechanisms and functions of the 5-hmC. This modification is suggested to be an intermediate between methylation and demethylation of the genome. Occupancy of 5-hmC correlates with inactive or non-productive promoters (1). 5-hmC pattern is different from 5-methylcytosine (5-mC) in many instances (2). Recently, a few analytical methods like chromatography coupled to mass spectroscopy and 5-hmC DNA immunoprecipitation have been developed to quantitate and analyze 5-hmC. However, all the methods fall short of acheiving quantitative site specific resolution.


  1. Bhutani N et al. (2011) Cell 146(6):866-72. PMID: 21925312
  2. Kinney SM et al. (2011) J Biol Chem. 286(28):24685-93. PMID: 21610077
  1. High Sensitivity 5-hmC Detection in Balb/C Brain Tissue

    Follow NEB Product Development Scientist, Romas Vaisvila, Ph.D., as he demonstrates the EpiMark® 5-mc and 5-hmc Analysis Kit for locus specific identification and quantification of 5-hmc in this protocol "High Sensitivity 5-hmc detection in Balb/C Brain Tissue".

  2. What Is Epigenetics?

    If all cells are created from the same genetic material, why are there so many different cell types? Listen to Sriharsa Pradhan, Senior Scientist, RNA Biology at NEB, as he describes how DNA is methylated and how this affects the path of reading the DNA code the same way an obstruction would derail a train off its tracks.

FAQs for 5-hmC Detection & Analysis

Protocols for 5-hmC Detection & Analysis

    Publications related to 5-hmC Detection & Analysis:

  1. Bhattacharyya, S., Yu, Y., Suzuki, M., Campbell, N., Mazdo, J., Vasanthakumar, A., et al. (2013). Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer Nucleic Acids Research. DOI: doi:10.1093/nar/gkt601

EpiMark® 5-hmC and 5-mC Analysis Kit Overview

Balb/C mouse tissue samples using the EpiMark 5-hmC and 5-mC Analysis Kit.
  1. Endpoint PCR of the 6 different reactions needed for methylation analysis. The boxed lanes indicate the presence of 5-hmC.
  2. Real time PCR data was used to determine amounts of 5-hmC and 5-mC present. The results demonstrate a variation in 5-hmC levels in the tissue sources indicated.