Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA

Tamar Shahal, Noa Gilat, Yael Michaeli, Orit Redy-Keisar, Doron Shabat, Yuval Ebenstein

Research output: Contribution to journalArticlepeer-review

Abstract

5-Hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine, is an important epigenetic mark linked to regulation of gene expression in development, and tumorigenesis. We have developed a spectroscopic method for a global quantification of 5hmC in genomic DNA. The assay is performed within a multiwell plate, which allows simultaneous recording of up to 350 samples. Our quantification procedure of 5hmC is direct, simple, and rapid. It relies on a two-step protocol that consists of enzymatic glucosylation of 5hmC with an azide-modified glucose, followed by a "click reaction" with an alkyne-fluorescent tag. The fluorescence intensity recorded from the DNA sample is proportional to its 5hmC content and can be quantified by a simple plate reader measurement. This labeling technique is specific and highly sensitive, allowing detection of 5hmC down to 0.002% of the total nucleotides. Our results reveal significant variations in the 5hmC content obtained from different mouse tissues, in agreement with previously reported data.

Original languageEnglish
Pages (from-to)8231-8237
Number of pages7
JournalAnalytical Chemistry
Volume86
Issue number16
DOIs
StatePublished - 19 Aug 2014

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