# US 12,601,006 B2 · Targeted, long-read nucleic acid sequencing for the determination of cytosine modifications > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,601,006 B2 - Document ID: US12601006B2 - Status: Granted - Kind: B2 - Application number: 17/929,033 - Filed: 2021-02-10 - Published: 2023-03-09 - Granted: 2026-04-14 - Earliest claimed: 2020-02-11 - Assignees: Ludwig Institute for Cancer Research - Applicants: Ludwig Institute for Cancer Research Ltd - Inventors: Chunxiao Song; Yibin Liu - Web page: https://patentgrep.com/patent/US12601006B2 ## CPC - C12Q 1/6827: for detection of mutation or polymorphism - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The method of claim 1, further comprising providing a quantitative level of 5mC or 5hmC at each location in the target nucleic acid using the percentages of a DHU or T at each transition location. ### Claim 3 (depends on 1) The method of claim 1, wherein the method further comprises inactivating the TET enzyme by providing a protease, by a change in temperature, and/or by a change in pH. ### Claim 4 (depends on 3) The method of claim 3, wherein the protease is proteinase K. ### Claim 5 (depends on 1) The method of claim 1, wherein the method further comprises the step of amplifying the copy number of the modified target nucleic acid. ### Claim 6 (depends on 1) The method of claim 1, wherein the borane reducing agent is selected from the group consisting of pyridine borane, 2-picoline borane (pic-BH3), borane, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride. ### Claim 7 (depends on 1) The method of claim 1, further comprising adding a glucose blocking group to the 5hmC in the nucleic acid sample prior to converting 5mC and/or 5hmC in the nucleic acid sample to 5caC and/or 5fC. ### Claim 8 (depends on 7) The method of claim 7, wherein the step of adding a glucose blocking group to the 5hmC is done in the same tube as the step of converting 5mC and/or 5hmC in the nucleic acid sample to 5caC and/or 5fC, and wherein the method is performed without a purification step between the step of adding a glucose blocking group to the 5hmC and the step of converting 5mC and/or 5hmC in the nucleic acid sample to 5caC and/or 5fC. ### Claim 9 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). ### Claim 10 (depends on 9) The method of claim 9, wherein said nucleic acid sample comprises less than or equal to about 100 ng of said whole genomic DNA. ### Claim 11 (depends on 9) The method of claim 9, wherein the whole genomic DNA comprises a plurality of unmodified cytosines. ### Claim 12 (depends on 9) The method of claim 9, wherein the method further comprises identifying the location of said 5mC or said 5hmC in said modified whole genomic DNA comprising detecting the presence of DHU in the modified whole genomic DNA, or converting the DHU to thymine (T) and detecting the presence of the thymine (T) in the modified whole genomic DNA; and wherein a cytosine (C) to DHU transition or a cytosine (C) to thymine (T) transition in the modified whole genomic DNA compared to the whole genomic DNA provides the location of a 5mC or 5hmC in the whole genomic DNA. ### Claim 13 (depends on 9) The method of claim 9, wherein the method further comprises converting said DHU to thymine (T).