Modification of DNA on magnetic beads

• US 10,704,083 B2
• Filed: 10/18/2018
• Issued: 07/07/2020
• Est. Priority Date: 01/30/2012
• Status: Active Grant

• Current Assignee: Exact Sciences Corporation
• Original Assignee: Exact Sciences Development Company LLC (Exact Sciences Corporation)
• Inventors: Domanico, Michael J., Allawi, Hatim, Lidgard, Graham P., Aizenstein, Brian, Hunt, Oliver, Zutz, Tobias Charles
• Primary Examiner(s): Crow, Robert T.
• Application Number:
  US16/164,373
• Publication Number:
  US 20190048399A1
• Time in Patent Office: 628 Days
• Field of Search: None
• 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 bisulfite 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 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/μ

l and 100 ng/μ

l 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.