Method for marking 5-formyl cytosine and use thereof in single base resolution sequencing

Patent metadata

Field	Value
Publication number	US-11293050-B2
Application number	US-201816489653-A
Country	US
Kind code	B2
Filing date	Feb 26, 2018
Priority date	Feb 28, 2017
Publication date	Apr 5, 2022
Grant date	Apr 5, 2022

Abstract

Disclosed are a method for marking 5-formyl cytosine and the use thereof in single base resolution sequencing. The method for marking the 5-formyl cytosine comprises the following steps of: (1) preparing a DNA or RNA sample; and (2) mixing the DNA or RNA sample with a buffer solution and a compound R1—CH2—CN to obtain a marking reaction system; and reacting the compound R1—CH2—CN therein with the 5-formyl cytosine in DNA and RNA molecules, and thereby achieving the marking of the 5-formyl cytosine; the reaction process is as in (I) below:wherein, R1 is an electron withdrawing group next to the CH2 group, preferably —CN, (II) or (III), and more preferably —CN; R is a DNA or RNA molecule connected to the 5-formyl cytosine; and the pH value of the marking reaction system is 7.5-9. On this basis, also provided in the present invention is a sequencing analysis method for the 5-formyl cytosine. The method can be implemented at a single cell level, and can achieve the sequencing of single-base resolution levels.

First claim

The invention claimed is:

1. A method for labeling 5-formyl cytosine, comprising:
(1) preparing a DNA or RNA sample;
(2) mixing the DNA or RNA sample with a buffer solution and a compound of formula R 1 —CH 2 —CN to obtain a labeling reaction system; and reacting the compound of formula R 1 —CH 2 —CN with 5-formyl cytosine in a DNA or RNA molecule to label 5-formyl cytosine according to the following reaction scheme:

wherein, R 1 is an electron withdrawing group adjacent to CH 2 ; R is a DNA or RNA molecule linked to 5-formyl cytosine; and the labeling reaction system has a pH of 7.5-9, wherein the concentration of the compound of formula R 1 —CH 2 —CN in the labeling reaction system is in the range from 75 mM to 1500 mM.

2. The method according to claim 1 , wherein the labeling reaction system has a pH of 8-9.

3. The method according to claim 1 , wherein in step (2), the reaction is performed at 20° C. to 60° C. for 12-48 hours.

4. A sequencing method of 5-formyl cytosine at single-base resolution, comprising:
(i) labeling a DNA or RNA sample by using the method according to claim 1 ;
(ii) amplifying and sequencing a labeling reaction system after completion of reaction to obtain a sequencing result of a labeled sample; and
(iii) comparing the sequencing result of the labeled sample with a reference sequence map of DNA or RNA, and determining a base at a certain position in the sample as 5-formyl cytosine if the base at the same position in the reference sequence map is read as cytosine and the base at the same position in the labeled sample is read as thymine.

5. The method according to claim 4 , wherein the DNA or RNA sample is a trace sample or a sample obtained from a single cell.

6. The method according to claim 4 , wherein the labeling reaction system after completion of the reaction in the step (ii) is directly subjected to amplification without purification.

7. The method according to claim 4 , wherein a method for amplification is a MALBAC or scRRBS amplification method.

8. An amplification system for DNA or RNA, comprising the labeling reaction system after completion of the reaction in step (ii) according to claim 4 .

9. A kit for sequencing 5-formyl cytosine at single-base resolution, comprising a buffer solution with a pH of 7.5-9, malononitrile and an amplification-related reagent, wherein the concentration of malononitrile in the labeling reaction system is in the range from 75 mM to 1500 mM.

10. A method for detecting 5-formyl cytosine quantitatively, comprising:
(a) sequencing N known pattern sequences with different content of 5-formyl cytosine by using the method according to claim 5 and determining a proportion of C-T conversion, wherein N≥2; the proportion of C-T conversion is a proportion of the number of bases read as cytosine C before labeling and read as thymine T after labeling at the same position in the sequence relative to the total number of the bases read as cytosine and thymine after labeling;
(b) plotting a standard curve with the content of 5-formyl cytosine as the horizontal/vertical coordinate and the proportion of C-T conversion as the vertical/horizontal coordinate;
(c) sequencing DNA or RNA with an unknown content of 5-formyl cytosine by using the method according to claim 5 , and determining the proportion of C-T conversion; and
(d) determining the content of 5-formyl cytosine in the DNA or RNA with the unknown content of 5-formyl cytosine, based on the proportion of C-T conversion determined in step (c) and the standard curve in step (b).

11. The method according to claim 1 , wherein, R 1 is —CN,

12. The method according to claim 1 , wherein the labeling reaction system has a pH of 8.

13. The method according to claim 1 , wherein the concentration of the compound of formula R 1 —CH 2 —CN in the labeling reaction system is in the range from 75 mM to 1000 mM.

14. The method according to claim 1 , wherein the concentration of the compound of formula R 1 —CH 2 —CN in the labeling reaction system is in the range from 75 mM to 500 mM.

15. The method according to claim 1 , wherein in step (2), the reaction is performed at 30° C. to 40° C.

16. The method according to claim 1 , wherein in step (2), the reaction is performed at 37° C.

17. The method according to claim 1 , wherein in step (2), the reaction is performed for 18-30 hours.

18. The method according to claim 1 , wherein in step (2), the reaction is performed for 20 hours.

19. The method according to claim 4 , wherein the single cell is derived from an embryonic stem cell, a gamete, an early embryo, a cancer cell, a nerve cell or a blood cell.

Assignees

• Univ Beijing

Inventors

• Yi Chengqi
• Zhu Chenxu
• Xia Bo

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C07H21/02 Primary

  with ribosyl as saccharide radical · CPC title