Method and materials for quaternary amine catalyzed bisulfite conversion of cytosine to uracil
View Patent ↗The invention provides methods and materials for the conversion of cytosine to uracil. A nucleic acid, such a gDNA, is reacted with bisulfate, such as magnesium bisulfite, in the presence of a quaternary amine catalyst. Examples of suitable quaternary amine catalysts include but are not limited to quaternary ammonium compounds, quaternary alkyl ammonium salts, quaternary alkyl ammonium halides, quaternary methyl ammonium bromide, quaternary ammonium chloride, tetraethylammonium hydroxide, tetraethylammonium chloride, tetrabutylammonium chloride, tetrabutylammonium bromide. The invention also contemplates kits of premeasured ingredients for carrying out the methods of the invention either on an individual sample or on a plurality of samples.
1. A method for converting cytosine to uracil comprising the steps of:
providing a nucleic acid comprising at least one cytosine nucleobase; and
reacting said nucleic acid with a bisulfite ion, in the presence of a quaternary amine catalyst having the Formula:
or a derivative thereof, wherein:
R 1 , R 2 , R 3 and R 4 are each independently C 1 -C 4 alkyl; and
Z is selected from halides and OH.
2. The method of claim 1 , wherein said quaternary amine catalyst comprises a quaternary alkyl ammonium compound.
3. The method of claim 1 , wherein said quaternary amine catalyst comprises a quaternary alkyl ammonium salt.
4. The method of claim 1 , wherein said quaternary amine catalyst comprises a quaternary alkyl ammonium halide.
5. The method of claim 1 , wherein said quaternary amine catalyst comprises a quaternary ammonium chloride or a quaternary ammonium bromide.
6. The method of claim 1 , wherein said quaternary amine catalyst comprises at least one of quaternary methyl ammonium bromide, tetraethylammonium hydroxide, tetraethylammonium chloride, tetrabutylammonium chloride and tetrabutylammonium bromide.
7. The method of claim 1 , wherein said catalyst comprises tetraethylammonium hydroxide.
8. The method of claim 1 , wherein said reaction is performed in a solution containing bisulfite ion in a concentration of from about 0.5M to about 2M.
9. The method of claim 8 , wherein said solution contains bisulfite ion in a concentration of about 1M to about 2M.
10. The method of claim 1 , wherein said reaction is performed in a solution containing magnesium bisulfite in a concentration of from about 0.5M to about 2M.
11. The method of claim 10 , wherein said solution contains magnesium bisulfite in a concentration of about 1M to about 2M.
12. The method of claim 5 , wherein said reaction is performed in a solution containing magnesium bisulfite in a concentration of from about 0.5M to about 2M.
13. The method of claim 7 , wherein said reaction is performed in a solution containing magnesium bisulfite in a concentration of from about 0.5M to about 2M.
14. The method of claim 3 , wherein said reaction is performed in a solution containing magnesium bisulfite in a concentration of from about 0.5M to about 2.5M.
15. The method of claim 1 , wherein said bisulfite solution comprises magnesium bisulfite having a concentration of at least about 1M.
16. A method for conversion of cytosine to uracil comprising the steps of:
reacting a DNA sample in solution with a bisulfite ion and quaternary amine catalyst having the Formula:
or a derivative thereof wherein:
R 1 , R 2 , R 3 and R 4 each independently C 1 -C 4 alkyl; and
Z is selected from halides and OH and,
wherein the final concentration of bisulfite is at least from about 1M to about 3M.
17. The method of claim 16 , wherein said final concentration of said bisulfite is about 1.3M.
18. The method of claim 16 , wherein said reaction is performed at about 50 degrees for about 4 to about 15 hours.
19. The method of claim 16 , wherein said bisulfite salt comprises magnesium bisulfite, and said quaternary amine catalyst comprises tetraethylaxnmonium hydroxide.
20. The method of claim 19 , wherein said reaction is performed at about 50 degrees for about 4 to about 15 hours.
21. The method of claim 1 , wherein said nucleic acid is gDNA.
22. The method of claim 1 , further comprising treating the product of said reaction of said nucleic acid and said bisulfite ion with a base.
23. The method of claim 22 , wherein said base is NaOH.