Isolation of nucleic acids

Patent metadata

Field	Value
Publication number	US-9631228-B2
Application number	US-201514713289-A
Country	US
Kind code	B2
Filing date	May 15, 2015
Priority date	May 12, 2011
Publication date	Apr 25, 2017
Grant date	Apr 25, 2017

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.

First claim

We claim:

1. A method for isolating a human target nucleic acid from a stool sample for use in a nucleic acid detection reaction, the method comprising:
a) removing an assay inhibitor from said sample to produce a clarified sample preparation, said removing comprising the steps of:
a1) homogenizing said sample to produce a homogenate;
a2) centrifuging said homogenate to produce a supernatant;
a3) treating said supernatant with an insoluble inhibitor-adsorbing composition to bind inhibitor, if present, in an insoluble inhibitor complex; and
a4) removing said insoluble inhibitor complex from said supernatant to produce a clarified sample preparation;

b) capturing said target nucleic acid from said clarified sample preparation, wherein capturing said target nucleic acid comprises:
b1) exposing said clarified sample preparation to a denaturing condition to produce a denatured sample;
b2) binding said target nucleic acid in said denatured sample to a target-specific capture reagent to form a capture complex;
b3) isolating said capture complex from the clarified sample preparation; and
b4) recovering said target nucleic acid from said capture complex in a target nucleic acid solution; and

c) performing a nucleic acid detection reaction on said target nucleic acid solution wherein at least one third of the volume of said nucleic acid detection reaction is from said target nucleic acid solution.

2. The method of claim 1 , further comprising:
d) retaining residual clarified sample preparation after said isolating said capture complex in step b3); and
e) repeating the capturing, isolating, and recovering of steps b1) to b4) using the residual clarified sample preparation and a second capture reagent that is specific for a second target nucleic acid.

3. The method of claim 1 , wherein said inhibitor-adsorbing composition is polyvinylpyrrolidone.

4. The method of claim 3 , wherein said polyvinylpyrrolidone is provided as a capsule or pressed tablet comprising a premeasured amount of polyvinylpyrrolidone.

5. The method of claim 1 , wherein isolating said inhibitor complex comprises centrifuging to separate said inhibitor complex from said supernatant.

6. The method of claim 5 , wherein during said centrifuging to separate said inhibitor complex from said supernatant, a fluid fraction of said supernatant passes through porous filtering material to produce said clarified sample preparation.

7. The method of claim 5 , wherein said isolating said inhibitor complex from said supernatant comprises:
a) placing said supernatant comprising said inhibitor complex into a spin filter comprising a hollow body and a bottom end that are both made from porous filtering material; and
b) centrifuging said spin filter;
wherein during said centrifuging said spin filter, a fluid fraction of said supernatant passes through porous filtering material of said spin filter to produce said clarified sample preparation.

8. The method of claim 1 , wherein said target-specific capture reagent comprises an oligonucleotide complementary to at least a portion of said target nucleic acid.

9. The method of claim 1 , wherein said target-specific capture reagent comprises a magnetic particle and wherein said isolating said capture complex comprises exposing said capture complex to a magnetic field.

10. The method of claim 1 , wherein recovering said target nucleic acid comprises eluting said target nucleic acid from said capture complex.

11. The method of claim 1 , wherein said nucleic acid detection reaction comprises use of at least one enzyme selected from the group consisting of a restriction endonuclease, a flap endonuclease, a reverse transcriptase, a ligase, an RNA polymerase, and a DNA polymerase.

12. The method of claim 11 , wherein said nucleic acid detection reaction comprises a polymerase chain reaction.

13. The method of claim 1 , wherein the target human DNA is from a gene associated with colorectal cancer and/or colorectal adenoma.

14. A method for isolating a target human DNA from a 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 denaturant to said clarified stool supernatant to produce a sample solution;
f) heating said sample solution;
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 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;
j) eluting the target human DNA, from the complex to produce a target nucleic acid solution comprising the target nucleic acid, when present; and
k) performing a nucleic acid detection reaction on said target nucleic acid solution wherein at least one third of the volume of said nucleic acid detection reaction is from said target nucleic acid solution.

15. The method of claim 14 , wherein said nucleic acid detection reaction comprises use of at least one enzyme selected from the group consisting of a restriction endonuclease, a flap endonuclease, a reverse transcriptase, a ligase, an RNA polymerase, and a DNA polymerase.

16. The method of claim 15 , wherein said nucleic acid detection reaction comprises a polymerase chain reaction.

17. The method of claim 14 , wherein the target human DNA is from a gene associated with colorectal cancer and/or colorectal adenoma.

Assignees

• Exact Sciences Corp

Inventors

• Bruinsma Janelle J
• Domanico Michael J
• Lidgard Graham P
• Zou Hongzhi
• Weisburg William G
• Shenoi Hemanth D
• Light Ii James P
• Kopitzke Keith
• Zeis John

Classifications

• C12Q2600/158

  Expression markers · CPC title

• Y10T436/143333

  Saccharide [e.g., DNA, etc.] · CPC title

• C12N15/1013

  by using magnetic beads · CPC title

• B01L3/5021 Primary

  Test tubes specially adapted for centrifugation purposes · CPC title

• B01D33/155

  the filtering surface being parallel to the rotation axis · CPC title