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 method for converting small DNA, the method comprising:
a) in a solution, combining single-stranded small DNA with a sulfonation reagent to produce treated small DNA comprising at least one of
sulfonated cytosine deoxynucleotides; and
5-methylcytosine deoxynucleotides,
wherein the small DNA is 200 or fewer nucleotides in length;
b) combining the treated small DNA in the solution of step a) with silica or silica-coated beads and an alcohol-free binding buffer comprising guanidine hydrochloride, incubating to produce bead-bound treated small DNA, and collecting the bead-bound treated small DNA from the alcohol-free binding buffer;
c) contacting collected bead-bound treated small DNA with a desulfonation reagent comprising NaOH and isopropanol to produce bead-bound converted small DNA comprising at least one of
uracil deoxynucleotides; and
5-methylcytosine deoxynucleotides,
and collecting the bead-bound converted small DNA from the desulfonation reagent;
d) eluting converted small DNA to provide an analytical sample comprising converted small DNA.
2. The method of claim 1 , wherein prior to step c), collected bead-bound treated small DNA is washed with a wash buffer.
3. The method of claim 2 , wherein the wash buffer is the alcohol-free binding buffer.
4. The method of claim 1 , wherein prior to step d), bead-bound converted small DNA is washed with a wash buffer comprising ethanol.
5. The method of claim 4 , wherein the wash buffer comprising ethanol comprises Tris hydrochloride buffer and 80% ethanol.
6. The method of claim 1 , wherein the sulfonation reagent comprises ammonium hydrogen sulfite.
7. The method of claim 1 , wherein the alcohol-free binding buffer comprises 6.5-7.5 M guanidine hydrochloride.
8. The method of claim 7 wherein the alcohol-free binding buffer comprises 7.0 M guanidine hydrochloride.
9. The method of claim 1 , wherein the desulfonation reagent comprises 60% to 70% isopropanol.
10. The method of claim 9 , wherein the desulfonation reagent comprises 70% isopropanol.
11. The method of claim 1 , wherein the desulfonation reagent comprises 0.075 to 0.125 N sodium hydroxide.
12. The method of claim 11 , wherein the desulfonation reagent comprises 0.1 N sodium hydroxide.
13. The method of claim 1 , wherein the method comprises a step of exposing bead-bound small DNA to a solution comprising at least one of bovine serum albumin or casein.
14. The method of claim 13 , wherein the solution comprising at least one of bovine serum albumin or casein comprises between 10 ng/μl and 100 ng/μl bovine serum albumin.
15. The method of claim 13 , wherein the solution comprising at least one of bovine serum albumin or casein comprises between 0.001% to about 0.01% casein.
16. The method of claim 1 , wherein the silica-coated beads are magnetic.