Detection of 5-hydroxymethylcytosine in genomic DNA
Diagnostic detection and precise mapping of 5-hydroxymethylcytosine modifications in genomic DNA via enzymatic digestion by PvuRts1I:
5-hydroxymethylcytosine (5-hmC) residues in the human genome have been implicated in the reprogramming of differentiated cells and are therefore potentially important markers for normal and pathologic development. The invention provides a method for determination of 5-hmC modifications with base-pair precision. Genomic DNA samples are melted into single stands, and the second strand synthesized with 5-hmC nucleotides, thus providing templates for cleavage with the endonuclease PvuRts1I. Cleavage products are identified by adapter ligation and either genome wide high-throughput sequencing for research applications, or quantitative PCR for residue specific diagnostic detection.
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