Modification of DNA on magnetic beads

• US 10,144,953 B2
• Filed: 03/11/2016
• Issued: 12/04/2018
• 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 THOMAS
• Application Number:
  US15/068,121
• Publication Number:
  US 20160265033A1
• Time in Patent Office: 998 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 bisulfate-converting small DNA, the method comprising:

a) in a solution combining single-stranded small DNA with a sulfonation reagent to produce sulfonated small DNA, wherein said small DNA is 200 or fewer bases in length;

b) combining the sulfonated small DNA in the solution of step a) with silica-coated magnetic beads and an alcohol-free binding buffer comprising guanidine hydrochloride and incubating to produce bead-bound sulfonated small DNA;

c) collecting bead-bound sulfonated small DNA from the binding buffer and contacting the collected bead-bound sulfonated small DNA with a desulfonation reagent comprising NaOH and isopropanol to produce bead-bound converted small DNA;

d) eluting converted small DNA to provide an analytical sample comprising converted small DNA.

2. The method of claim 1, wherein said alcohol-free binding buffer comprises about 7.0 M guanidine hydrochloride.

3. The method of claim 1, wherein said desulfonation reagent comprises about 60% to 70% isopropanol.

4. The method of claim 3, wherein said desulfonation reagent comprises about 70% isopropanol.

5. The method of claim 1, wherein said desulfonation reagent comprises about 0.075 to 0.125 N sodium hydroxide.

6. The method of claim 5, wherein the desulfonation reagent comprises about 0.1 N sodium hydroxide.

7. The method of claim 1, wherein said method comprises a step of exposing bead-bound small DNA to a solution comprising at least one of bovine serum albumin or casein.

8. The method of claim 7, wherein said solution comprising at least one of bovine serum albumin or casein comprises between about 10 ng/μ

l and 100 ng/μ

l bovine serum albumin.

9. The method of claim 7, wherein said solution comprising at least one of bovine serum albumin or casein comprises between about 0.001% to about 0.01% casein.