Bisulfite method
View Patent ↗The invention provides methods for purifying bisulfite-treated nucleic acid samples.
1. A method for the purification of a bisulfite-treated nucleic acid comprising the steps of:
(a) providing a liquid comprising a bisulfite-treated nucleic acid in a size-exclusion device comprising a porous, size-discriminating barrier;
(b) washing said nucleic acid in said size-exclusion device by passing liquid through said barrier while said imcleic acid is retained by said barrier;
(c) treating said washed nucleic acid with a base in said size-exclusion device to increase the pH; and
(d) optionally washing said nucleic acid resulting from step (c), resulting in a purified bisulfite-treated nucleic acid.
2. The method of claim 1 wherein said step (d) is performed in said size-exclusion device.
3. The method of claim 1 wherein said porous, size-discriminating barrier has a 300K cut off.
4. The method of claim 1 wherein said porous, size-discriminating barrier has a 30K cut off.
5. The method of claim 1 wherein said porous, size-discriminating barrier has a 100K cut off.
6. A method of converting one or more cytosine bases to uracil bases in a nucleic acid, the method comprising:
contacting the nucleic acid with a bisulfite conversion reagent, thereby forming a bisulfite-treated nucleic acid,
washing the treated nucleic acid in a size-exclusion device comprising a porous, size-discriminating barrier that allows residual bisulfite conversion reagent to pass through while retaining the bisulfitetreated nucleic acid, and
subjecting the retained bisulfite-treated nucleic acid to basic conditions comprising a pH of 10 or above, during or after said washing, to form a nucleic acid in which at least one cytosine base has been converted to a uracil base.
7. The method of claim 6 wherein the porous, size-discrimination barrier comprises a membrane.
8. The method of claim 6 wherein said barrier has a 300K cut off.
9. The method of claim 6 wherein said barrier has a 100K cut off.
10. The method of claim 6 wherein said barrier has a 30K cut off.
11. The method of claim 6 wherein said washing is performed under pressure.
12. The method of claim 6 wherein said washing comprises centrifugation of the device.
13. The method of claim 6 wherein said bisulfite conversion reagent comprises sodium bisulfite.
14. The method of claim 6 wherein said bisulfite conversion reagent comprises magnesium bisulfite.
15. The method of claim 6 wherein said basic conditions comprise 0.1M NaOH.