Spectroscopic Quantification of Global 5’Hydroxymethylcytosine in Genomic DNA

Tamar Shahal, School of Chemistry, Tel Aviv University, Tel Aviv, Israel
Yuval Ebenstein, School of Chemistry, Tel Aviv University, Tel Aviv, Israel

The information content of  DNA extends beyond the genetic base composition in the form of dynamic chemical modifications such as methylation and hydroxymethylation. These epigenetic DNA modifications are subject to change during normal development as well as pathogenesis. For example, 5-hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine, is an important epigenetic mark, linked to development, gene transcription regulation and tumorigenesis. We have developed a spectroscopic method for global quantification of 5hmC in genomic DNA. The assay is performed on a multiwell plate, allowing the simultaneous recording of up to 350 samples. Our 5hmC quantification procedure is direct, simple and rapid. It relies on a two-step protocol consisting of an enzymatic glucosylation of 5hmC with an azide-modified glucose followed by a click reaction with a fluorescently tagged alkyne. 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 a detection limit down to 0.002% 5hmC to total nucleotides. Our results show significant variations in the 5hmC content of different mouse tissues, in agreement with previously reported data.