Isolation of nucleic acids

Patent metadata

Field	Value
Publication number	US-9845491-B2
Application number	US-201514939925-A
Country	US
Kind code	B2
Filing date	Nov 12, 2015
Priority date	May 12, 2011
Publication date	Dec 19, 2017
Grant date	Dec 19, 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 composition comprising a residual supernatant from a human stool homogenate comprising human DNA and bacterial DNA, said residual supernatant produced by a method comprising:
i) preparing a stool supernatant fluid from said human stool homogenate,
ii) treating the stool supernatant fluid with an insoluble inhibitor adsorbant comprising polyvinylpolypyrrolidone particles to remove assay inhibitors to produce a treated stool supernatant fluid,
iii) exposing the treated stool supernatant fluid to a plurality of different magnetic target-specific capture particles, wherein different target-specific capture particles comprise covalently-attached oligonucleotides complementary to at least a portion of different target human DNAs, wherein said different target human DNAs comprise at least two target DNAs selected from NDRG4, BMP3, and KRAS, under conditions wherein said plurality of different target-specific capture reagents form target-specific capture reagent/target human DNA complexes, and
iv) removing target-specific capture reagent/target human DNA complexes from said treated stool supernatant fluid to produce said residual supernatant, wherein said residual supernatant is treated stool supernatant fluid from which the target-specific capture reagent/target human DNA complexes have been removed, said residual supernatant comprising bacterial DNA and human DNAs not complementary to the oligonucleotides of the target-specific capture particles used in step iii).

2. The composition of claim 1 , wherein said different target human DNAs comprise NDRG4, BMP3, and KRAS.

3. The composition of claim 1 , wherein said different target human DNAs further comprise a reference gene.

4. The composition of claim 3 , wherein said reference gene is β-actin.

5. A composition comprising a residual supernatant from a human stool homogenate comprising human DNA and bacterial DNA, said residual supernatant produced by a method comprising:
i) preparing a stool supernatant fluid from said human stool homogenate, wherein preparing a stool supernatant fluid comprises
a) homogenizing a human stool sample in a buffer to produce a human stool homogenate;
b) partitioning solids from fluid in the human stool homogenate of step a) and collecting the fluid as a stool supernatant fluid;

ii) treating the stool supernatant fluid with an insoluble inhibitor adsorbent comprising polyvinylpolypyrrolidone particles to remove assay inhibitors, wherein polyvinylpolypyrrolidone particles bound to assay inhibitors are removed from said stool supernatant fluid by filtration to produce treated stool supernatant fluid;
iii) adding a chaotropic agent to the treated stool supernatant fluid of step ii) and exposing the treated stool supernatant fluid to a plurality of different magnetic target-specific capture particles,
wherein different target-specific capture particles comprise covalently-attached oligonucleotides complementary to at least a portion of different target human DNAs, wherein said different target human DNAs comprise at least two target DNAs selected from NDRG4, BMP3, and KRAS,
under conditions wherein said plurality of different target-specific capture reagents form target-specific capture reagent/target human DNA complexes, and

iv) removing target-specific capture reagent/target human DNA complexes from said treated stool supernatant fluid of step iii) to produce said residual supernatant, wherein said residual supernatant is treated stool supernatant fluid from which the target-specific capture reagent/target human DNA complexes have been removed, said residual supernatant comprising bacterial DNA and human DNAs not complementary to the oligonucleotides of the target-specific capture particles used in step iii).

6. The composition of claim 5 , wherein the target-specific capture particles are magnetic particles.

7. The composition of claim 5 , wherein said human stool homogenate comprises at least 4 grams of stool from a human subject.

8. The composition of claim 5 , wherein the chaotropic agent comprises guanidine thiocyanate.

9. The composition of claim 8 , wherein said guanidine thiocyanate is present at a concentration of 2-3 M.

10. The composition of claim 5 , wherein said different target human DNAs comprise NDRG4, BMP3, and KRAS.

11. The composition of claim 5 , wherein said different target human DNAs further comprise a reference gene.

12. The composition of claim 11 , wherein said reference gene is β-actin.

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

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12N15/1006 Primary

  by means of a solid support carrier, e.g. particles, polymers · CPC title

• C12N15/1017

  by filtration, e.g. using filters, frits, membranes · CPC title

• Y10T436/143333

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

• B04B3/00

  Centrifuges with rotary bowls in which solid particles or bodies become separated by centrifugal force and simultaneous sifting or filtering · CPC title