MODIFICATION OF DNA ON MAGNETIC BEADS

• US 20130196322A1
• Filed: 01/30/2013
• Published: 08/01/2013
• Est. Priority Date: 01/30/2012
• Status: Active Grant

• Current Assignee: Exact Sciences Corporation
• Original Assignee: Exact Sciences Corporation
• Inventors: Domanico, Michael J., Allawi, Hatim, Lidgard, Graham P., Aizenstein, Brian, Hunt, Oliver, Zutz, Tobias Charles
• Granted Patent: US 9,315,853 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2523/113 Denaturating agents
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2600/154 Methylation markers

Abstract

Provided herein is technology related to the chemical modification and purification of DNA. Specifically, the technology provides methods for performing a bisulfate conversion reaction on small amounts of single-stranded, fragmented DNA and performing the subsequent desulfonation and purification steps on magnetic beads.

Claims

1. A method for treating DNA to quantify DNA methylation, the method comprising contacting a DNA with a sulfonation reagent to produce sulfonated DNA, and binding said sulfonated DNA to a magnetic bead in an alcohol-free binding buffer.

2. The method of claim 1, wherein said DNA is contacted with said sulfonation reagent to produce a mixture comprising sulfonated DNA, and wherein said mixture is combined with said magnetic bead in the presence of said alcohol-free binding buffer.

3. The method of claim 1 wherein the sulfonation reagent is ammonium hydrogen sulfite.

4. The method of claim 1 wherein the alcohol-free binding buffer comprises guanidine hydrochloride.

5. The method of claim 1 wherein the binding buffer comprises approximately 7.0 M guanidine hydrochloride.

6. The method of claim 1 wherein the DNA consists of 200 or fewer bases in length.

7. The method of claim 1 wherein the DNA is single stranded.

8. The method of claim 1 further comprising contacting sulfonated DNA bound to said bead with a desulfonation reagent to produce converted DNA, and eluting said converted DNA to provide an analytical sample.

9. The method of claim 8 wherein the amount of DNA contacted with said sulfonation reagent is substantially the same as the amount of DNA in the analytical sample.

10. The method of claim 8 wherein a cytosine, if present in the DNA prior to sulfonation, is converted to a uracil in the DNA in said converted DNA

11. The method of claim 8 wherein a methylcytosine, if present in the DNA prior to sulfonation, is not converted to a uracil in said converted DNA.

12. The method of claim 1 wherein the magnetic bead is a silica-coated magnetic bead.

13. The method of claim 8, wherein the process of producing said converted DNA is completed in 4 hours or less.

14. The method of claim 8, wherein the process of producing said converted DNA is completed in 3 hours or less.

15. The method of claim 8, wherein the process of producing said converted DNA is completed in 2 hours or less.

16. The method of claim 1, wherein the desulfonation reagent comprises isopropanol.

17. The method of claim 1, wherein the desulfonation reagent comprises approximately 70% isopropanol and approximately 0.1 N sodium hydroxide.

18. The method of claim 1, wherein said magnetic bead and said DNA are in a sample vessel, and wherein said method comprises a step of exposing said sample vessel containing said magnetic bead and said DNA to a solution comprising at least one of bovine serum albumin or casein.

19. The method of claim 18, wherein said solution comprises between about 10 ng/μ

l and 100 ng/μ

l bovine serum albumin.

20. The method of claim 18 wherein said solution comprises between about 0.001% to about 0.01% casein.

21. A kit for treating DNA comprising:

a) an ammonium hydrogen sulfite sulfonation reagent;

b) a magnetic bead;

c) a guanidine hydrochloride alcohol-free binding buffer;

d) a wash buffer;

e) desulfonation reagent; and

f) an elution buffer.

22. The kit of claim 21 wherein the binding buffer comprises approximately 6.5-7.5 M guanidine hydrochloride.

23. The kit of claim 21, wherein the desulfonation reagent comprises isopropanol.

24. The method of claim 23, wherein the desulfonation reagent comprises approximately 70% isopropanol and approximately 0.1 N sodium hydroxide.

25. The kit of claim 21, further comprising a solution containing at least one of bovine serum albumin and/or casein.