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US 2014/0194608 A1Published application
Isolation of nucleic acids
AssigneeExact Sciences Corporation
InventorsJanelle J. Bruinsma, Michael J. Domanico, Graham P. Lidgard, Hongzhi Zou, William G. Weisburg, Hemanth D. Shenoi, James P. Light, II, Keith Kopitzke, John Zeis
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Abstract
Provided herein is technology relating to isolating nucleic acids. In particular, the technology relates to methods and kits for extracting nucleic acids from problematic samples such as stool.
Claims21
A system for isolating a target human DNA from a human stool sample, the system comprising:
- a) a volume of stool homogenization solution suitable for processing a human stool sample having a mass of at least 4 grams;
- and
- b) a target-specific capture reagent comprising an oligonucleotide covalently attached to a magnetic particle, wherein said oligonucleotide is complementary to at least a portion of said target human DNA
- c) a spin filter, comprising
- i) a hollow body;
- ii) a bottom end;
- and
- iii) an open top end opposite the bottom end,
- wherein the hollow body of said spin filter is made from a porous filtering material, preferably a polyethylene porous filtering material, and wherein the bottom end is optionally made from said porous filtering material.
6 more elements
A method for isolating a target human DNA from a human stool sample, the method comprising:
- a) obtaining a stool sample having a mass of at least 4 grams from a human subject;
- b) homogenizing said stool sample in an homogenization buffer to produce an homogenized stool sample;
- c) preparing a stool supernatant from the homogenized stool sample;
- d) treating said stool supernatant with PVP to produce a clarified stool supernatant;
- e) adding guanidine thiocyanate to 10 milliliters of clarified stool supernatant to produce a sample solution comprising 2-3 M guanidine thiocyanate;
- f) heating said sample solution to 90° C. for 10 minutes;
- g) adding to said sample solution a target-specific capture reagent comprising an oligonucleotide covalently attached to a magnetic particle, wherein said oligonucleotide is complementary to at least a portion of said target human DNA;
- h) incubating said sample solution with said target-specific capture reagent at ambient temperature for approximately 1 hour to produce a complex comprising said target-specific capture reagent and said target human DNA;
- i) exposing the sample solution comprising said complex to a magnetic field to isolate the complex from the sample solution, and retaining the sample solution;
- j) eluting the target human DNA, from the complex to produce a target nucleic acid solution comprising the target nucleic acid, when present;
- k) repeating steps f-j of the method using a different target-specific capture reagent in each step g, to produce a different target nucleic acid solution in each step j;
- and
- l) performing a nucleic acid detection reaction on each different target nucleic acid solution, wherein at least one third of the volume of each said nucleic acid detection reaction is from said target nucleic acid solution.
10 more elements
Dependencies are inferred from the claim text.