Isolation of nucleic acids

Patent metadata

Field	Value
Publication number	US-10047390-B2
Application number	US-201715807970-A
Country	US
Kind code	B2
Filing date	Nov 9, 2017
Priority date	May 12, 2011
Publication date	Aug 14, 2018
Grant date	Aug 14, 2018

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 DNA from a fluid fraction prepared from human stool homogenate for use in a nucleic acid detection reaction, the method comprising:
i) treating a fluid fraction from a human stool homogenate with polyvinylpyrrolidone to bind inhibitor, if present, in an insoluble PVP inhibitor complex, to produce a treated stool fluid;
ii) exposing the treated stool fluid to a target sequence-specific capture reagent comprising particles covalently attached to oligonucleotides complementary to at least a portion of human target DNA, under conditions wherein the target sequence-specific capture reagent forms a target sequence-specific capture reagent/human target DNA capture complex,
iii) separating the capture complex from said treated stool fluid;
iv) recovering human target DNA from the capture complex in a human target DNA solution; and
v) performing a DNA detection reaction on said human target DNA solution.

2. The method of claim 1 , further comprising:
vi) retaining residual treated stool fluid after said separating said capture complex in iii); and
vii) repeating the exposing, separating, and recovering of steps ii) to iv) using the residual treated stool fluid and a second target sequence-specific capture reagent that is specific for a second human target DNA.

3. The method of claim 1 , wherein said insoluble PVP inhibitor complex comprises polyvinylpolypyrrolidone.

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

5. The method of claim 1 , wherein treating said fluid fraction from a human stool homogenate with PVP to produce said treated stool fluid comprises passing treated stool fluid through porous filtering material, wherein the insoluble PVP inhibitor complex is retained by the porous filtering material.

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

7. The method of claim 1 , wherein recovering said human target DNA comprises eluting said human target DNA from said capture complex.

8. 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.

9. The method of claim 8 , wherein said nucleic acid detection reaction comprises a polymerase chain reaction.

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

• C12N15/1013

  by using magnetic beads · CPC title

• B01L2300/0681

  Filter · CPC title

• B01D33/15

  with rotary plane filtering surfaces · CPC title

• C12N15/1017

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

• Y10T436/143333

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