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 the conversion of cytosine bases in a DNA molecule to uracil bases, comprising:
a) binding a double stranded DNA directly to an unmodified solid phase without a compound mediating the binding to the solid phase, wherein the unmodified solid phase is selected from the group consisting of glass, silica, and a glass or silica containing material,
b) denaturing the double stranded DNA,
c) incubating the solid phase bound DNA in the presence of sulfite ions whereby the DNA is deaminated, yielding a solid phase bound deaminated DNA,
d) optionally washing the solid phase bound deaminated DNA,
e) incubating the solid phase bound deaminated DNA under alkaline conditions whereby the deaminated DNA is desulfonated,
f) washing the solid phase bound deaminated and desulfonated DNA, and
g) eluting the washed deaminated and desulfonated DNA from the solid phase.
2. A method for conversion of cytosine bases in a DNA molecule to uracil bases comprising:
a) binding a double stranded DNA directly to an unmodified solid phase without a compound mediating the binding to the solid phase, wherein the unmodified solid phase is selected from the group consisting of glass, silica, and glass or silica containing material,
b) denaturing the double stranded DNA,
c) incubating the solid phase bound DNA in the presence of sulfite ions whereby the DNA is deaminated, yielding a solid phase bound deaminated DNA,
d) washing the solid phase bound deaminated DNA,
e) eluting the washed deaminated DNA from the solid phase, and
f) incubating the deaminated DNA under alkaline conditions whereby the deaminated DNA is desulfonated.
3. The method according to claim 1 or 2 wherein the solid phase is a glass fleece or a glass membrane.
4. The method according to claim 1 or 2 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 4 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 the sol-gel method.
9. The method according to claim 8 , wherein said sol-gel method comprises:
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 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 of claim 1 , wherein the denaturing in method step b) is performed in the presence of NaOH.
11. A method for the conversion of cytosine bases in a DNA molecule to uracil bases, comprising:
a) denaturing a double stranded DNA,
b) incubating denatured double stranded DNA in the presence of sulfite ions whereby the DNA is deaminated, yielding a deaminated DNA,
c) binding the deaminated double stranded DNA directly to an unmodified solid phase without a compound mediating the binding to the solid phase, wherein the unmodified solid phase is selected from the group consisting of glass, silica, and a glass or silica containing material,
d) optionally, washing the solid phase bound deaminated DNA,
e) incubating the solid phase bound deaminated DNA under alkaline conditions whereby the deaminated DNA is desulfonated,
f) washing the solid phase bound deaminated and desulfonated DNA, and
g) eluting the washed deaminated and desulfonated DNA from the solid phase.