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US 10,704,083 B2Granted
Modification of DNA on magnetic beads
AssigneeExact Sciences Development Company, LLC
InventorsMichael J. Domanico, Hatim Allawi, Graham P. Lidgard, Brian Aizenstein, Oliver Hunt, Tobias Charles Zutz
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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.
Claims16
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.
9 more elements
Dependencies are inferred from the claim text.