TY - JOUR
T1 - Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA
AU - Shahal, Tamar
AU - Gilat, Noa
AU - Michaeli, Yael
AU - Redy-Keisar, Orit
AU - Shabat, Doron
AU - Ebenstein, Yuval
PY - 2014/8/19
Y1 - 2014/8/19
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84906264992&partnerID=8YFLogxK
U2 - 10.1021/ac501609d
DO - 10.1021/ac501609d
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C2 - 25072105
AN - SCOPUS:84906264992
SN - 0003-2700
VL - 86
SP - 8231
EP - 8237
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 16
ER -