title: Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA.
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.
authors: Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University , Tel Aviv, 6997801, Israel.
authors: Tamar
authors: T
authors: Shahal
authors: Noa
authors: N
authors: Gilat
authors: Yael
authors: Y
authors: Michaeli
authors: Orit
authors: O
authors: Redy-Keisar
authors: Doron
authors: D
authors: Shabat
authors: Yuval
authors: Y
authors: Ebenstein
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D004271:DNA, Fungal / Q000737:chemistry; D004867:Equipment Design; D023281:Genomics / Q000295:instrumentation*; D057230:Limit of Detection; D051379:Mice; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
chemical_list: D004271:DNA, Fungal; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA