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US 2021/0317519 A1Published application
Bisulfite-free, base-resolution identification of cytosine modifications
AssigneeLudwig Institute for Cancer Research
InventorsChunxiao Song, Yibin Liu
Life of this patent
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Abstract
This disclosure provides methods for bisulfite-free identification in a nucleic acid sequence of the locations of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxylcytosine and 5-formylcytosine.
Claims20
A method for identifying 5mC or 5hmC in a target nucleic acid comprising the steps of:
- providing a nucleic acid sample comprising the target nucleic acid;
- modifying the nucleic acid comprising the steps of:
- converting the 5mC and 5hmC in the nucleic acid sample to 5-carboxylcytosine (5caC) and/or 5fC;
- and
- converting the 5caC and/or 5fC to dihydrouracil (DHU) by treating the nucleic acid with a borane reducing agent to provide a modified nucleic acid sample comprising a modified target nucleic acid;
- and
- detecting the sequence of the modified target nucleic acid;
- wherein a cytosine (C) to thymine (T) transition or a cytosine (C) to DHU transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides the location of either a 5mC or 5hmC in the target nucleic acid.
5 more elements
A method for identifying 5mC or 5hmC in a target nucleic acid comprising the steps of:
- providing a nucleic acid sample comprising the target nucleic acid;
- modifying the nucleic acid comprising the steps of:
- converting the 5mC and 5hmC in the nucleic acid sample to 5-carboxylcytosine (5caC) and/or 5fC by contacting the nucleic acid sample with a TET enzyme so that the one or more 5caC or 5fC residues are generated;
- and
- converting the 5caC and/or 5fC to dihydrouracil (DHU) by treating the nucleic acid with a borane reducing agent to provide a modified nucleic acid sample comprising a modified target nucleic acid;
- and
- detecting the sequence of the modified target nucleic acid;
- wherein a cytosine (C) to thymine (T) transition or a cytosine (C) to DHU transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides the location of either a 5mC or 5hmC in the target nucleic acid.
5 more elements
Dependencies are inferred from the claim text.