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.
Claims125
A spin filter comprising
- a) a hollow body;
- b) a bottom end;
- and
- c) an open top end opposite the bottom end,
- wherein the hollow body is made from a porous filtering material.
A method for removing an assay inhibitor from a crude sample preparation comprising a nucleic acid, the method comprising:
- a) 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 complex;
- b) separating the complex from the crude sample preparation to produce a clarified sample preparation comprising said nucleic acid.
A system for removing an assay inhibitor from a crude sample preparation comprising a nucleic acid, the system comprising:
- a) insoluble polyvinylpyrrolidone for binding the assay inhibitor and producing a complex;
- b) functionality for separating the complex from the crude sample preparation to produce a clarified sample preparation comprising said nucleic acid;
- and
- c) functionality for retaining a clarified sample preparation comprising the nucleic acid.
A method for isolating a plurality of different target nucleic acids from a stool sample, the method comprising:
- a) contacting a stool sample preparation comprising a plurality of different target nucleic acids with a first target-specific capture reagent under conditions wherein a first target nucleic acid binds to said first target-specific capture reagent to form a first complex;
- b) separating said first complex from said stool sample to produce a first residual stool sample preparation;
- c) contacting said first residual stool sample preparation with a second target-specific capture reagent under conditions wherein a second target nucleic acid binds to said second target-specific capture reagent to form a second complex;
- d) separating said second complex from said first residual stool sample to produce a second residual stool sample preparation;
- e) eluting said first target nucleic acid from said first complex to produce a first target nucleic acid solution;
- and
- f) eluting said second target nucleic acid from said second complex to produce a second target nucleic acid solution.
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.
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;
- 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.
A kit for preparing a nucleic acid solution from a sample, the kit comprising:
- a) polyvinylpyrrolidone for removing an inhibitor from the sample;
- b) a reagent for capturing a target nucleic acid from the sample;
- and
- c) a functionality for producing a magnetic field.
A kit for removing an assay inhibitor from a crude sample preparation comprising a nucleic acid, the kit comprising:
- a) insoluble polyvinylpyrrolidone;
- and
- b) a spin column.
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;
- 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.
A system for preparing a nucleic acid solution from a sample, the system comprising:
- a) polyvinylpyrrolidone for removing an inhibitor from the sample;
- b) a reagent for capturing a target nucleic acid from the sample;
- and
- c) a functionality for producing a magnetic field.
A system for removing an assay inhibitor from a crude sample preparation comprising a nucleic acid, the system comprising:
- a) insoluble polyvinylpyrrolidone;
- and
- b) a spin column.
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.
Dependencies are inferred from the claim text.