Method for bisulfite treatment
The present application is directed to a method for performing a bisulfite reaction to determine methylation positions in a nucleic acid, i.e. methylated and non-methylated cytosines, whereby the nucleic acid is bound to a solid phase during the deamination and/or desulfonation step of the bisulfite reaction. The solid phase is preferably a material comprising glass or silica, more preferably a glass fleece, glass membrane or a magnetic glass particle. Further, the use of a solid phase for binding a nucleic acid during the deamination and/or desulfonation step of the bisulfite reaction is disclosed and a kit containing a bisulfite reagent and a solid phase.
1. A method for conversion of a cytosine base in a nucleic acid to a uracil base, comprising the steps of:
a. incubating the nucleic acid in the presence of sulfite ions whereby the nucleic acid is deaminated;
b. binding the deaminated nucleic acid to a solid phase;
c. optionally washing the deaminated solid phase bound nucleic acid;
d. incubating the deaminated solid phase bound nucleic acid under alkaline conditions whereby the deaminated nucleic acid is desulfonated, thereby converting the cytosine base in the nucleic acid to the uracil base;
e. washing the deaminated and desulfonated solid phase bound nucleic acid; and
f. eluting the washed deaminated and desulfonated nucleic acid from the solid phase.
2. The method according to claim 1 , wherein the solid phase is a material comprising silica or glass.
3. The method according to claim 1 , wherein the solid phase is a glass fleece or a glass membrane.
4. The method according to claim 1 , wherein the solid phase is a magnetic glass particle.
5. The method according to claim 4 , wherein the magnetic glass particle has a mean diameter between 0.5 μm and 5 μm.
6. The method according to claim 4 , wherein the magnetic glass particle contains a magnetic object with a diameter between 5 and 500 nm.
7. The method according to claim 6 , wherein the magnetic glass particle contains a magnetic object with a mean diameter of 23 nm.
8. The method according to claim 4 , wherein the magnetic glass particle is manufactured by a sol-gel method.
9. The method of claim 8 , wherein said sol-gel method comprises the steps of
a. suspending magnetic objects in a sol,
b. hydrolyzing the sol to cover the magnetic objects with a gel,
c. spray-drying the magnetic objects covered with a gel with a powder in a two-nozzle spray-drier, and
d. sintering the spray-dried powder to form a glass from the gel covering the magnetic objects.
10. The method according to claim 1 , wherein the solid phase contains pores.
11. The method according to claim 10 , wherein the nucleic acid is bound to surfaces in said pores.