Modification of DNA on magnetic beads
First Claim
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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.
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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 bisulfate conversion reaction on small amounts of single-stranded, fragmented DNA and performing the subsequent desulfonation and purification steps on magnetic beads.
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Citations
8 Claims
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1. A method for treating DNA to quantify DNA methylation in fragmented, small DNA, the method comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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Specification