Modification of DNA on magnetic beads

• US 9,315,853 B2
• Filed: 01/30/2013
• Issued: 04/19/2016
• 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
• Primary Examiner(s): CROW, ROBERT THOMAS
• Application Number:
  US13/754,631
• Publication Number:
  US 20130196322A1
• Time in Patent Office: 1,175 Days
• US Class Current: 1/1
• 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 in fragmented, small DNA, the method comprising:

a) to a sample solution comprising small DNA, adding NaOH to produce a solution comprising strands of single-stranded small DNA;

b) to said solution comprising strands of single-stranded small DNA, adding a sulfonation reagent comprising ammonium hydrogen sulfite to produce a mixture of small DNA in a solution comprising ammonium hydrogen sulfite, and incubating the mixture at a temperature of 65°

C. to 68°

C. for 2 hours or less to produce sulfonated small DNA;

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

d) collecting bead-bound 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;

e) eluting converted small DNA to provide an analytical sample comprising converted small DNA; and

f) quantifying DNA methylation by identifying or enumerating methylated cytosines, if present, in the converted small DNA in the analytical sample;

wherein said small DNA is 200 or fewer bases in length and wherein the amount of small DNA in the analytical sample is at least 90% of the amount of small DNA contacted with the sulfonation reagent.

2. The method of claim 1 wherein the alcohol-free binding buffer comprises approximately 7.0 M guanidine hydrochloride.

3. The method of claim 1, wherein the mixture of small DNA in ammonium hydrogen sulfite is incubated for 1 hour.

4. The method of claim 3, wherein the mixture of small DNA in ammonium hydrogen sulfite is incubated at 66°

C.

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

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

7. The method of claim 6, wherein said solution comprises between about 10 ng/μ

l and 100 ng/μ

l bovine serum albumin.

8. The method of claim 6 wherein said solution comprises between about 0.001% to about 0.01% casein.