Bisulfite-free, base-resolution identification of cytosine modifications

Patent metadata

Field	Value
Publication number	US-11306355-B2
Application number	US-202117201825-A
Country	US
Kind code	B2
Filing date	Mar 15, 2021
Priority date	Jan 8, 2018
Publication date	Apr 19, 2022
Grant date	Apr 19, 2022

Abstract

This disclosure provides methods for bisulfite-free identification in a nucleic acid sequence of the locations of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxylcytosine and 5-formylcytosine.

First claim

The invention claimed is:

1. A method for identifying 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) in a target nucleic acid comprising the steps of:
providing a nucleic acid sample comprising the target nucleic acid;
modifying the target nucleic acid comprising the steps of:
converting the 5mC and 5hmC in the nucleic acid sample to 5-carboxylcytosine (5caC) and/or 5-formylcytosine (5fC) by contacting the nucleic acid sample with a ten-eleven translocation (TET) enzyme so that one or more 5caC or 5fC residues are generated; and
converting the 5caC and/or 5fC to dihydrouracil (DHU) by treating the target nucleic acid with a borane reducing agent to provide a modified nucleic acid sample comprising a modified target nucleic acid; and

detecting the sequence of the modified target nucleic acid;

wherein a cytosine (C) to thymine (T) transition or a cytosine (C) to DHU transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides the location of either a 5mC or 5hmC in the target nucleic acid.

2. The method of claim 1 , wherein the borane reducing agent is 2-picoline borane.

3. The method of claim 1 , wherein the step of detecting the sequence of the modified target nucleic acid comprises one or more of chain termination sequencing, microarray, high-throughput sequencing, and restriction enzyme analysis.

4. The method of claim 1 , wherein the TET enzyme is selected from the group consisting of human TET1, TET2, and TET3; murine Tet1, Tet2, and Tet3; Naegleria TET (NgTET); and Coprinopsis cinerea (CcTET).

5. The method of claim 1 , further comprising a step of blocking one or more modified cytosines.

6. The method of claim 5 , wherein the step of blocking comprises adding a sugar to a 5hmC.

7. The method of claim 1 , wherein the method further comprises a step of amplifying the copy number of one or more nucleic acid sequences.

8. A method for chemically modifying a nucleic acid sample comprising the steps of:
providing a nucleic acid sample comprising 5-carboxylcytosine (5caC) and/or 5-formylcytosine (5fC); and
converting the 5caC and/or 5fC to dihydrouracil (DHU) by treating the nucleic acid with a borane reducing agent to provide a modified nucleic acid sample comprising a modified nucleic acid.

9. The method of claim 8 , wherein the borane reducing agent is selected from the group consisting of 2-picoline borane (pic-BH3), borane, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.

10. The method of claim 8 , wherein the borane reducing agent is 2-picoline borane.

11. The method of claim 8 , further comprising the step of detecting the sequence of the modified nucleic acid.

12. The method of claim 11 , wherein the step of detecting the sequence of the modified nucleic acid comprises one or more of chain termination sequencing, microarray, high-throughput sequencing, and restriction enzyme analysis.

13. The method of claim 11 , wherein the sequencing step provides a quantitative level of one or more cytosine modifications in the nucleic acid sample.

14. The method of claim 8 , wherein the method further comprises a step of amplifying the copy number of one or more nucleic acid sequences.

Assignees

• Ludwig Inst For Cancer Res Ltd

Inventors

• Song Chunxiao
• Liu Yibin

Classifications

• C12Y204/01027

  DNA beta-glucosyltransferase (2.4.1.27) · CPC title

• C12Y204/01026

  DNA alpha-glucosyltransferase (2.4.1.26) · CPC title

• C12Q2600/154

  Methylation markers · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title