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AssigneeBluestar Genomics, Inc.
InventorsPatrick A. Arensdorf, Damek Spacek, Chunxiao Song
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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-carboxymethylcytosine 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.
A method for converting oxidized 5-methylcytosine residues in cell-free DNA to dihydrouracil residues, comprising contacting cell-free DNA containing at least one oxidized 5-methylcytosine residue selected from 5-carboxymethylcytosine, 5-formylcytosine, and combinations thereof with an organic borane effective to reduce, deaminate, and either decarboxylate or deformylate the at least one oxidized 5-methylcytosine residue, thereby providing a dihydrouracil residue in place thereof.
A reaction mixture comprising:
A method for detecting the presence and location of 5-methylcytosine residues in cell-free DNA, wherein the method comprises:
A kit for converting 5-methylcytosine residues in cell-free DNA to dihydrouracil residues, comprising a blocking reagent composition for blocking the 5-hydroxymethylcytosine residues, an oxidizing reagent to oxidize the 5-methylcytosine residues beyond hydroxymethylation to provide oxidized 5-methylcytosine residues, and an organic borane effective to reduce, deaminate, and either decarboxylate or deformylate the oxidized 5-methylcytosine residues.
A kit for identifying 5-methylcytosine residues in a cell-free DNA sample, comprising individual reagent compositions for modifying 5-hydroxymethylcytosine residues to provide an affinity tag thereon, removing the modified 5-hydroxymethylcytosine residues from the sample, and oxidizing unmodified 5-methylcytosine residues beyond hydroxymethylation to provide oxidized 5-methylcytosine residues, and an organic borane effective to reduce, deaminate, and either decarboxylate or deformylate the oxidized 5-methylcytosine residues.
A method for detecting the presence and location of 5-methylcytosine residues and 5-hydroxymethylcytosine residues in cell-free DNA, wherein the method comprises:
A method for identifying a co-occurrence of 5-methylcytosine and 5-hydroxymethylcytosine on a single DNA strand in cell-free DNA, comprising:
A method for detecting the presence and location of 5-methylcytosine residues and 5-hydroxymethylcytosine residues in a DNA sample, wherein the method comprises:
Dependencies are inferred from the claim text.