Modification of DNA on magnetic beads
Provided herein is technology related to the chemical modification and purification of DNA. Specifically, the technology provides methods for performing a bisulfite conversion reaction on small amounts of single-stranded, fragmented DNA and performing the subsequent desulfonation and purification steps on magnetic beads.
1. A system comprising:
a) a composition comprising:
i) single-stranded small DNA comprising sulfonated cytosine deoxynucleotides, wherein the small DNA is 200 or fewer nucleotides in length, and wherein the small DNA is bound to silica or silica-coated beads, in a mixture with
ii) an alcohol-free solution comprising ammonium hydrogen sulfite and 5.5 to 7.5 M guanidine hydrochloride;
b) a desulfonation reagent comprising NaOH and isopropanol; and
c) a wash buffer comprising ethanol.
2. The system of claim 1 , further comprising a solution comprising at least one of bovine serum albumin and/or casein.
3. The system of claim 2 , wherein the solution comprises between 10 ng/μ1 and 100 ng/μ1 bovine serum albumin.
4. The system of claim 2 , wherein the solution comprises between 0.001% to about 0.01% casein.
5. The system of claim 1 , wherein the alcohol-free solution comprises 6.5-7.0 M guanidine hydrochloride.
6. The system of claim 1 , wherein the alcohol-free solution comprises 7.0 M guanidine hydrochloride.
7. The system of claim 1 , wherein the desulfonation reagent comprises 60% to 70% isopropanol.
8. The system of claim 1 , wherein the desulfonation reagent comprises 70% isopropanol.
9. The system of claim 1 , wherein the desulfonation reagent comprises 0.075 to 0.125 N sodium hydroxide.
10. The system of claim 1 , wherein the desulfonation reagent comprises 0.1 N sodium hydroxide.
11. The system of claim 1 , wherein the silica-coated beads are magnetic.
12. The system of claim 11 , further comprising a magnet.
13. The system of claim 1 , wherein the composition comprises 6.6 to 6.8% ammonium hydrogen sulfite.