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US 2014/0194607 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.
Claims27
A kit for isolating a target human DNA from a human stool sample, the kit comprising:
- a) a volume of stool homogenization solution suitable for processing a human stool sample having a mass of at least 4 grams;
- b) a spin column comprising 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 is made from a porous filtering material
- and
- c) 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.
6 more elements
A method for isolating a target nucleic acid from a sample, the method comprising:
- a) removing an assay inhibitor from said sample to produce a clarified sample preparation, wherein said removing an assay inhibitor comprises
- i) adding insoluble polyvinylpyrrolidone to said crude sample preparation prior to isolating the nucleic acid under conditions wherein said assay inhibitor binds to said polyvinylpyrrolidone to produce a polyvinylpyrrolidone-assay inhibitor complex in a sample to be filtered;
- ii) separating the complex from the crude sample preparation to produce a clarified sample preparation comprising said nucleic acid, wherein said separating comprises placing said sample to be filtered into a spin filter, said spin filter comprising
- 1) a hollow body;
- 2) a bottom end;
- and
- 3) an open top end opposite the bottom end,
- wherein the hollow body 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, and
- centrifuging said spin filter, wherein, during said centrifuging, said polyvinylpyrrolidone-assay inhibitor complex is retained in said spin filter and filtrate passed through said porous filtering material is a clarified sample preparation;
- b) capturing said target nucleic acid from said clarified sample preparation with a target-specific capture reagent to form a capture complex;
- c) isolating said capture complex from the clarified sample preparation;
- and
- d) recovering said target nucleic acid from said capture complex in a nucleic acid solution.
10 more elements
Dependencies are inferred from the claim text.