DNA Methylation and HydroxymethylationTools & Technology

Shining a Light on 5-hmC: Direct Labeling and Optical Detection of DNA Hydroxymethylation

dna hydroxymethylation

DNA hydroxymethylation is a DNA modification that has been known to exist for decades, but its importance to biology and its role as an epigenetic mark have only been appreciated for the past few years.  The presence of 5-hydroxymethylcytosine (5-hmC) has been observed in the genomes of many different organisms, and in mammals its levels are the highest in the brain and other tissues of the central nervous system. Because the interest in 5-hmC as a novel epigenetic mark has increased in recent years, there has also been an increase in the development of tools and technologies to investigate this DNA modification.  Dr. Yuval Ebenstein’s research group at Tel Aviv University recently published an article describing a new method that they developed to investigate 5-hmC levels in DNA using an innovative optical detection approach.

In their recent report, Michaeli et al. expand upon previous studies that use the T4 bacteriophage β-glucosyltranfserase enzyme to specifically label 5-hydroxymethycytosine residues with glucose moieties modified to contain a functional azide group.  Other groups have used this approach to biotinylate the glucosylated 5-hmC residues using “click” chemistry, but researchers in Dr. Ebenstein’s lab instead added a fluorescent molecule, allowing direct detection of 5-hmC in DNA using either microscopy or NanoDrop® spectrophotometers (or any other UV-Vis spectrophotometer with the ability to read multiple wavelengths).  Detection of the fluorescently labeled 5-hmC residues using microscopy combined with modified glass slides and microfluidics allows single-molecule investigation of DNA hydroxymethylation, and importantly is also compatible with simultaneous detection of other epigenetic components (e.g. histones and DNA methylation) and DNA binding proteins (e.g. RNA polymerases and transcription factors).  Using a UV-Vis spectrophotometer, such as a NanoDrop®, this method allows researchers to very quickly, inexpensively, and sensitively measure the global levels of DNA hydroxymethylation, in even small amounts of DNA.

This sensitive new method developed by Ebenstein’s group should lend itself well for detection of the low levels of 5-hmC that are present in most tissues, and also allows renewed interrogation of tissues and organisms previously thought to lack the 5-hmC modification.  Additionally, Dr. Ebenstein has recently used this method to investigate the changes in DNA hydroxymethylation that occur over the course of embryonic stem cell development and differentiation.  How do you think this new method can be applied to your research project?


Michaeli Y, Shahal T, Torchinsky D, Grunwald A, Hoch R, & Ebenstein Y (2013). Optical detection of epigenetic marks: sensitive quantification and direct imaging of individual hydroxymethylcytosine bases. Chemical communications (Cambridge, England) PMID: 23756466

(image credit: Yuval Ebenstein)

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Kevin B.

Kevin B.

Kevin grew up in Northern California and has also spent several years living on the East Coast. When he is not in the lab, Kevin enjoys snowboarding, watching NFL games (and playing fantasy football!), spending time outdoors, and exploring Southern California.