# US 2014/0194608 A1 · Isolation of nucleic acids

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- Number: US 2014/0194608 A1
- Document ID: US20140194608A1
- Status: Published application
- Kind: A1
- Application number: 14/145,082
- 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/US20140194608A1

## 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 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
c) 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 of said spin filter 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.

### Claim 2 (depends on 1)

The system of claim 1, further comprising a magnet.

### Claim 3 (depends on 1)

The system of claim 1, further comprising a polyvinylpyrrolidone.

### Claim 4 (depends on 3)

The system of claim 3, wherein said polyvinylpyrrolidone is a polyvinylpolypyrrolidone.

### Claim 5 (depends on 1)

The system of claim 1 wherein said polyvinylpyrrolidone is in a premeasured form.

### Claim 6 (depends on 3)

The system of claim 3 wherein said polyvinylpyrrolidone is provided as a tablet.

### Claim 7 (depends on 1)

The system of claim 1 further comprising guanidine thiocyanate.

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 9)

The system of claim 9, 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 11 (depends on 1)

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

### Claim 12 (depends on 11)

The system of claim 11, 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 13 (depends on 11)

The system of claim 11, further comprising a sample container configured to hold said stool sample.

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 1)

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

### Claim 16 (depends on 1)

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

### Claim 17 (depends on 16)

The system of claim 16, wherein said control comprises a nucleic acid.

### Claim 18

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.

### Claim 19 (depends on 18)

The method of claim 18, wherein the target human DNA is from a gene associated with a disease state selected from the group consisting of colorectal cancer and colorectal adenoma.

### Claim 20 (depends on 18)

The method of claim 18 wherein the repeating step k) is performed n times to produce n+1 different target nucleic acid solutions.

### Claim 21 (depends on 20)

The method of claim 20 wherein n is at least 3.