Methods for the epigenetic analysis of DNA, particularly cell-free DNA
AssigneeBluestar Genomics, Inc.
InventorsPatrick A. Arensdorf, Damek Spacek
Life of this patent
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Abstract
Methods are provided for the epigenetic analysis of cell-free DNA using organic boranes to convert oxidized 5-methylcytosine residues in the cell-free DNA to dihydrouracil (DHU) residues. Cell-free DNA is contacted with an organic borane selected to successively bring about reduction, deamination, and decarboxylation of oxidized 5-methylcytosine residues such as 5-carboxylcytosine and 5-formylcytosine, resulting in DHU residues in place thereof. Following amplification, the treated cell-free DNA is sequenced, with the DHU residues read as thymine residues. Reaction mixtures, kits and additional methods are also provided, as are related methods for the epigenetic analysis of DNA, including cell-free DNA.
Claims11
A method for detecting the locations of 5mC and 5hmC residues in a cell-free DNA sample comprising fragmented, adapter-ligated template DNA, wherein the method comprises:
- in a first fraction of the sample,
- (a) blocking 5hmC residues with a blocking reagent to yield blocked 5hmC residues;
- (b) enzymatically oxidizing 5mC residues to provide oxidized 5mC residues selected from 5caC, 5fC, and combinations thereof;
- (c) converting the oxidized 5mC residues to DHU by treatment with pyridine borane, thereby providing first fraction DNA comprising blocked 5hmC residues and DHU at 5mC locations;
- and
- (d) amplifying and sequencing the first fraction DNA to provide first fraction sequence reads in which the blocked 5hmC residues read as C and DHU reads as T;
- and in a second fraction of the sample,
- (e) oxidizing 5hmC residues with an oxidizing reagent effective to convert 5hmC residues to oxidized 5hmC residues without modifying 5mC residues, wherein the oxidized 5hmC residues are selected from 5caC, 5fC, and combinations thereof;
- and
- (f) converting the oxidized 5hmC residues to DHU by treatment with pyridine borane, thereby providing second fraction DNA comprising unmodified 5mC residues and DHU in place of the oxidized 5hmC residues;
- (g) amplifying and sequencing the second fraction DNA to provide second fraction sequence reads in which the unmodified 5mC residues read as C and DHU reads as T;
- and
- (h) comparing the first fraction sequence reads with the second fraction sequence reads to identify 5mC and 5hmC locations in the template DNA.
Dependencies are inferred from the claim text.