Bisulfite-free, base-resolution identification of cytosine modifications
View Patent ↗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.
1. A method comprising steps of:
Providing a nucleic acid sample comprising one or more 5-carboxylcytosine (5caC) or 5-formylcytosine (5fC) residues; and
Contacting the sample with a borane reducing agent under conditions that reduce 5caC and 5fC to dihydrouracil (DHU),
wherein the nucleic acid sample comprising one or more 5caC or 5fC residues comprises ligated nucleic acids.
2. The method of claim 1 , wherein the step of providing comprises:
Preparing the nucleic acid sample by contacting a target nucleic acid comprising one or more 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) residues with a Ten Eleven Translocation (TET) enzyme so that the one or more 5caC or 5fC residues are generated.
3. The method of claim 1 , wherein the reducing agent is selected from the group consisting of pyridine borane, 2-picoline borane (pic-BH 3 ), borane, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.
4. The method of claim 1 , wherein the reducing agent is pyridine borane or 2-picoline borane.
5. The method of claim 1 , further comprising a sequencing step.
6. The method of claim 5 , wherein the sequencing step distinguishes modified and unmodified cytosine in the nucleic acid sample.
7. The method of claim 5 , wherein the sequencing step provides a quantitative level of one or more cytosine modifications in the nucleic acid sample.
8. The method of claim 5 , wherein the sequencing step comprises one or more of chain termination sequencing, microarray, high-throughput sequencing, and restriction enzyme analysis.
9. The method of claim 1 , further comprising a step of blocking one or more modified cytosines.
10. The method of claim 9 , wherein the step of blocking comprises adding a sugar to a 5hmC.
11. The method of claim 10 , wherein the sugar is glucose or a modified glucose.
12. The method of claim 1 , wherein the step of providing comprises:
Preparing the nucleic acid sample by contacting a target nucleic acid comprising one or more 5hmC residues with a chemical oxidizing agent so that one or more 5fC residues are generated.
13. The method of claim 9 , wherein the step of blocking comprises contacting a nucleic acid sample comprising one or more 5fC residues with an aldehyde reactive compound selected from hydroxylamine derivatives, hydrazine derivatives, and hydrazide derivatives.
14. The method of claim 13 , wherein the hydroxylamine derivative is O-ethylhydroxylamine.
15. The method of claim 9 , wherein the step of blocking comprises contacting a nucleic acid sample comprising one or more 5caC residues with:
a carboxylic acid derivatization reagent; and
an amine, hydrazine, or hydroxylamine compound.
16. The method of claim 15 , wherein the step of blocking comprises contacting a nucleic acid sample comprising one or more 5caC residues with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and with ethylamine.
17. The method of claim 12 , wherein the oxidizing agent is potassium perruthenate or Cu(II)/2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO).
18. The method of claim 1 , wherein the method further comprises a step of amplifying the copy number of one or more nucleic acid sequences.
19. The method of claim 1 , wherein the ligated nucleic acids comprise an adapter sequence.