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US 12,601,006 B2Granted
Targeted, long-read nucleic acid sequencing for the determination of cytosine modifications
AssigneeLudwig Institute for Cancer Research
InventorsChunxiao Song, Yibin Liu
Life of this patent
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Abstract
The present disclosure provides a bisulfite-free, long-read, base-resolution method named long-read TAPS (lrTAPS) for detecting 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in a nucleic acid sequence. lrTAPS comprises mild enzymatic and chemical reactions to detect 5mC and 5hmC, the two major epigenetic marks found in the mammalian genome, quantitatively at base-resolution without affecting unmodified cytosine.
Claims13
A method for chemically modifying a nucleic acid sample, comprising the steps of:
- (a) converting 5-methyl cytosine (5mC) and/or 5-hydroxymethylcytosine (5hmC) in a nucleic acid sample comprising a target nucleic acid to 5-carboxylcytosine (5caC) and/or 5-formylcytosine (5fC) with a TET enzyme;
- and
- (b) converting 5caC and/or 5fC in the nucleic acid sample to dihydrouracil (DHU) with a borane reducing reagent to provide a modified nucleic acid sample comprising a modified target nucleic acid;
- wherein steps (a) and (b) are performed in a single reaction vessel without a purification step between steps (a) and (b);
- and wherein the modified target nucleic acid has a length of from 3 kb to 10 kb.
2 more elements
A method for determining a whole genome methylome, comprising the steps of:
- (a) converting 5-methyl cytosine (5mC) and/or 5-hydroxymethylcytosine (5hmC) in a nucleic acid sample comprising a whole genomic DNA of a cell to 5-carboxylcytosine (5caC) and/or 5-formylcytosine (5fC) with a ten eleven translocation (TET) enzyme;
- and
- (b) converting 5caC and/or 5fC in the nucleic acid sample to dihydrouracil (DHU) with a borane reducing reagent to provide a modified whole genomic DNA;
- and
- (c) amplifying and sequencing target nucleic acid sequences ranging from 3 kb to 10 kb in length;
- wherein steps (a) and (b) are performed in a single reaction vessel without a purification step between steps (a) and (b).
3 more elements
Dependencies are inferred from the claim text.