Bisulfite-free, base-resolution identification of cytosine modifications
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 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.