Granted Patent
B2
US 12,410,467 · App. 17/625,500 · Granted Sep 9, 2025
Bisulfite-free, whole genome methylation analysis
Inventors:
Chunxiao Song (Oxford, GB); Jingfei Cheng (Oxford, GB); Paulina Siejka-Zielinska (Oxford, GB); Yibin Liu (Oxford, GB)
Assignee:
LUDWIG INSTITUTE FOR CANCER RESEARCH LTD
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Abstract
This disclosure provides methods for cost-effective bisulfite-free identification in DNA, including whole genomic DNA, of the locations of one or more of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxylcytosine and 5-formylcytosine. The methods described herein are based on the conversion of modified cytosine (5mC, 5hmC, 5fC, 5caC) to dihydrouracil (DHU), for example by TET-assisted pyridine borane treatment, followed by endonuclease cleavage of the DHU, and identification of the cleavage site, which corresponds to the location of the modified cytosine.
Claims (12)
1. A method for cleaving a modified target DNA, the method comprising:
converting 5-carboxylcytosine (5caC) and/or 5-formylcytosine (5fC) in a target DNA to dihydrouracil (DHU) with a borane reducing agent to provide a modified target DNA comprising one or more DHU; and
contacting the modified target DNA comprising one or more DHU with one or more DHU-sensitive endonucleases that cleave the modified target DNA at, or adjacent to, the one or more DHU.
2. 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.
3. The method of claim 1 , wherein the one or more DHU-sensitive endonucleases are selected from one or more of the group consisting of a mixture of a combination of Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII, Endonuclease III, Tma Endonuclease III, Endonuclease VIII, and Formamidopyrimidine DNA Glycosylase (Fpg).
4. The method of claim 1 , wherein the one or more DHU-sensitive endonucleases comprise a mixture of a combination of UDG and Endonuclease VIII.
5. The method of claim 1 , further comprising the step of adding adapter DNA molecules to the cleaved modified target DNA.
6. The method of claim 1 , further comprising detecting the sequence of the cleaved modified target DNA, wherein the presence of a cleavage site provides the location of either a 5caC or 5fC in the target DNA.
7. 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; and
the one or more DHU-sensitive endonucleases are selected from one or more of the group consisting of a mixture of a combination of Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII, Endonuclease III, Tma Endonuclease III, Endonuclease VIII, and Formamidopyrimidine DNA Glycosylase (Fpg).
8. The method of claim 7 , wherein the one or more DHU-sensitive endonucleases comprise a mixture of a combination of UDG and Endonuclease VIII.
Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Sep 26, 2022
From: CHENG, JINGFEI; SONG, CHUNXIAO; LIU, YIBIN; SIEJKA-ZIELINSKA, PAULINA
To: THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
Reel/Frame 061209/0839 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Sep 26, 2022
From: THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
To: LUDWIG INSTITUTE FOR CANCER RESEARCH LTD
Reel/Frame 061209/0882 →
Continuity (2)
Provisional Application
62871444
· Jul 8, 2019
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