# US 2014/0194607 A1 · Isolation of nucleic acids

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- Number: US 2014/0194607 A1
- Document ID: US20140194607A1
- Status: Published application
- Kind: A1
- Application number: 14/145,070
- Filed: 2013-12-31
- Published: 2014-07-10
- Earliest claimed: 2011-05-11
- Assignees: Exact Sciences Corporation
- Applicants: Exact Sciences Corporation
- Inventors: Janelle J. Bruinsma; Michael J. Domanico; Graham P. Lidgard; Hongzhi Zou; William G. Weisburg; Hemanth D. Shenoi; James P. Light, II; Keith Kopitzke; John Zeis
- Web page: https://patentgrep.com/patent/US20140194607A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- B01D 33/15: with rotary plane filtering surfaces
- B01D 33/155: {the filtering surface being parallel to the rotation axis}
- B01L 3/00: Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L 3/5021: {Test tubes specially adapted for centrifugation purposes}
- B03C 1/30: Combinations with other devices, not otherwise provided for
- B04B 3/00: Centrifuges with rotary bowls in which solid particles or bodies become separated by centrifugal force and simultaneous sifting or filtering
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12N 15/1013: {by using magnetic beads}
- C12N 15/1017: {by filtration, e.g. using filters, frits, membranes}
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B03C: MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS (separating isotopes B01D59/00; combinations of magnetic or electrostatic separation with separation of solids by other means B03B, B07B)
- B04B: CENTRIFUGES (high-speed drum mills B02C19/11; domestic spin driers D06F; analysing, measuring or monitoring physical or chemical properties of samples during centrifuging, see the relevant subclasses for these procedures, e.g. G01N)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## 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.

## Claims

### Claim 1

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.

### Claim 2 (depends on 1)

The kit of claim 1, wherein the bottom end of said spin filter is made from a porous filtering material.

### Claim 3 (depends on 1)

The kit of claim 1, wherein the porous filtering material is polyethylene.

### Claim 4 (depends on 1)

The kit of claim 1, wherein the porous filtering material has a nominal pore size of 20 micrometers.

### Claim 5 (depends on 1)

The kit of claim 1, wherein the bottom end of said spin filter has a shape selected from the group consisting of a hemisphere, a disc, a cone, or a portion of an ellipsoid.

### Claim 6 (depends on 1)

The kit of claim 1, further comprising a magnet.

### Claim 7 (depends on 1)

The kit of claim 1, further comprising polyvinylpyrrolidone.

### Claim 8 (depends on 7)

The kit of claim 7 wherein the polyvinylpyrrolidone is a polyvinylpolypyrrolidone.

### Claim 9 (depends on 7)

The kit of claim 7 wherein the polyvinylpyrrolidone is in a premeasured form.

### Claim 10 (depends on 9)

The kit of claim 9 wherein the polyvinylpyrrolidone is provided as a tablet.

### Claim 11 (depends on 1)

The kit of claim 1 further comprising guanidine thiocyanate.

### Claim 12 (depends on 1)

The kit of claim 1, further comprising an elution or wash solution.

### Claim 13 (depends on 1)

The kit of claim 1, further comprising an apparatus to produce a magnetic field.

### Claim 14 (depends on 13)

The kit of claim 13, wherein said apparatus to produce a magnetic field comprises a first magnetic feature and a second magnetic feature, wherein a north pole of the first magnetic feature is placed in close proximity to the sample and a south pole of the second magnetic feature is placed in close proximity to the sample.

### Claim 15 (depends on 1)

The kit of claim 1, further comprising a device for collecting a stool sample.

### Claim 16 (depends on 15)

The kit of claim 15, wherein said device comprises:
a) a body;
b) a detachable sample capsule configured to move between an open state and a closed state, wherein the detachable sample capsule in said closed state comprises a sample collection space configured to enclose a sample; and
c) an ejector mechanically connected to said body, said ejector configured to detach said detachable sample capsule from said body when said detachable sample capsule is in a closed state.

### Claim 17 (depends on 1)

The kit of claim 1, further comprising a sample container configured to hold said stool sample.

### Claim 18 (depends on 1)

The kit of claim 1 further comprising a vessel in which to hold isolated target human DNA.

### Claim 19 (depends on 1)

The kit of claim 1, further comprising a shipping container.

### Claim 20 (depends on 1)

The kit of claim 1, further comprising a control reagent.

### Claim 21 (depends on 20)

The kit of claim 20, wherein the control comprises a nucleic acid.

### Claim 22

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.

### Claim 23 (depends on 22)

The method of claim 22, further comprising:
e) retaining residual clarified sample preparation after said isolating step;
f) repeating the capturing, isolating, and recovering steps using the residual clarified sample and a second capture reagent.

### Claim 24 (depends on 22)

The method of claim 22 wherein removing said assay inhibitor from said sample comprises:
a1) homogenizing said sample to produce a homogenate;
a2) centrifuging said homogenate to produce a supernatant.

### Claim 25 (depends on 22)

The method of claim 22, wherein said polyvinylpyrrolidone is a polyvinylpolypyrrolidone.

### Claim 26 (depends on 22)

The method of claim 22, wherein said polyvinylpyrrolidone is provided in a premeasured form.

### Claim 27 (depends on 22)

The method of claim 22, wherein said polyvinylpyrrolidone is provided as a tablet.