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Abstract

This disclosure describes, among other things, compositions, kits and methods for identifying to 5-hydroxymethylcytosine (5hmC) in eukaryotic genomic DNA. The compositions and methods utilize a PvuRts1I family enzyme for digesting eukaryotic genomic DNA suspected of containing 5hmC or 5-formylcytosine (5fC) modified nucleotides providing an end of the DNA with a two base overhang suitable for ligating an adapter at a fixed distance 3′ from an 5hmC. Random fragmentation of the genomic DNA can generate a blunt end suitable for attaching a second adapter. The DNA can be sequenced and 5hmC and 5fC residues identified and located at a defined position in the eukaryotic genome. An enrichment step may be used that utilizes a chemoselective agent capable of being selectively added to DNA containing 5hmC. An example is a glucosyltransferase and a glucosyltransferase substrate comprising a chemo-selective group. This DNA can then be enriched by immobilization on a matrix that binds the chemoselective agent added to the DNA containing 5hmC and DNA that does not contain a chemo-selective group is removed by washing. Adapters can be added at one or both ends of the restriction endonuclease cleaved DNA after random fragmentation prior to or after an enrichment step.

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