Method for bisulfite treatment
View Patent ↗The present application is directed to a method for performing a bisulfite reaction to determine methylation positions in a nucleic acid via treatment of the solid phase-bound nucleic acid with bisulfite, desulfonation and elution of the nucleic acid from the solid phase.
1. A method for the conversion of a cytosine base in a nucleic acid to an uracil base comprising the steps of
a1) binding the nucleic acid to a solid phase capable of binding nucleic acids and incubating the solid phase bound nucleic acid in the presence of sulfite ions thereby deaminating the cytosine base in the nucleic acid, or
a2) incubating the nucleic acid in the presence of sulfite ions thereby deaminating the cytosine base in the nucleic acid and binding the nucleic acid to a solid phase,
b) optionally washing the solid phase bound nucleic acid,
c) contacting the solid phase bound nucleic acid with a solution with an alkaline pH thereby desulfonating the base deaminated in step a1) or a2) in the nucleic acid and converting it to an uracil base and simultaneously eluting the nucleic acid.
2. The method according to claim 1 wherein the solution with an alkaline pH comprises NaOH or KOH.
3. The method according to claim 1 , wherein the solution with an alkaline pH comprises 10 to 100 mM NaOH or KOH.
4. The method according to claim 3 , wherein the solution with an alkaline pH comprises 20 to 60 mM NaOH or KOH.
5. The method according to claim 1 , wherein the temperature in step c) of the method of claim 1 is between 60 to 80 ° C.
6. The method according to claim 1 , wherein the solid phase is a material comprising silica or glass.
7. The method according to claim 1 , wherein the solid phase is a glass fleece or a glass membrane.
8. The method according to claim 1 , wherein the solid phase is a magnetic glass particle.
9. The method of claim 8 wherein the magnetic glass particle has a mean diameter between 0.5 μm and 5 μm.
10. The method according to claim 8 wherein the magnetic glass particle contains a magnetic object with a diameter between 5 and 500 nm.
11. A method for the amplification of a nucleic acid comprising the steps of
a1) binding the nucleic acid to a solid phase capable of binding nucleic acids and incubating the solid phase bound nucleic acid in the presence of sulfite ions thereby deaminating the cytosine base in the nucleic acid, or
a2) incubating the nucleic acid in the presence of sulfite ions thereby deaminating the cytosine base in the nucleic acid and binding the nucleic acid to a solid phase,
b) optionally washing the solid phase bound nucleic acid,
c) contacting the solid phase bound nucleic acid with a solution with an alkaline pH thereby
desulfonating the base deaminated in step a1) or a2) in the nucleic acid and converting it to an uracil base and
simultaneously eluting the nucleic acid,
d) amplifying the eluted nucleic acid by PCR or related method.
12. A kit for performing a bisulfite reaction comprising
a bisulfite salt or a solution comprising bisulfite ions,
a solid phase capable of binding nucleic acids, and
a solution comprising 10 to 100 mM NaOH or KOH.
13. The kit according to claim 12 wherein the solid phase is a material comprising silica or glass.
14. The kit according to claim 12 , wherein the solid phase is a glass fleece or a glass membrane.
15. The kit according to claim 12 , wherein the solid phase is a magnetic glass particle.
16. The kit according to claim 12 , wherein the solution of NaOH or KOH comprises 20 to 60 mM of NaOH or KOH.