(12) United States Patent
`Bruinsma et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 9,163,278 B2
`*Oct. 20, 2015
`
`US009 163278B2
`
`(54) ISOLATION OF NUCLEICACIDS
`
`(56)
`
`(71) Applicant: Exact Sciences Corporation, Madison,
`WI (US)
`
`(72) Inventors: Janelle J. Bruinsma, Madison, WI (US);
`Michael J. Domanico, Middleton, WI
`(US); Graham P. Lidgard, Madison, WI
`(US); Hongzhi Zou, Middleton, WI
`(US); William G. Weisburg, San Diego,
`CA (US); Hemanth D. Shenoi, Verona,
`WI (US); James P. Light, II, Middleton,
`WI (US); Keith Kopitzke, Fallbrook,
`CA (US); John Zeis, San Marcos, CA
`(US)
`
`(*) Notice:
`
`(73) Assignee: EXACT SCIENCES
`CORPORATION, Madison, WI (US)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`This patent is Subject to a terminal dis-
`claimer.
`
`(21) Appl. No.: 14/145,082
`
`22) Filed:
`(22) File
`
`Dec. 31, 2013
`ec. 5,
`
`(65)
`
`Prior Publication Data
`US 2014/01.94608A1
`Jul. 10, 2014
`
`References Cited
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`
`Related U.S. Application Data
`(63) Continuation
`of
`application
`PCT/US2012/037581, filed on May 11, 2012.
`(60) Provisional application No. 61/485,338, filed on May
`12, 2011, provisional application No. 61/485,386,
`filed on May 12, 2011, provisional application No.
`61/485.448, filed on May 12, 2011, provisional
`application No. 61/485,214, filed on May 12, 2011.
`
`No.
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`(51) Int. Cl.
`CI2O I/68
`BO3C I/30
`BOID 33/5
`BOIL 3/00
`CI2N IS/10
`B04B 3/00
`(52) U.S. Cl.
`CPC .............. CI2O I/6806 (2013.01); B0ID 33/15
`(2013.01); B0ID 33/155 (2013.01); B0IL3/00
`(2013.01); B0IL3/5021 (2013.01); B03C I/30
`(2013.01); B04B 3/00 (2013.01); C12N
`15/1006 (2013.01); Y10T 436/143333 (2015.01)
`(58) Field of Classification Search
`None
`See application file for complete search history.
`
`FOREIGN PATENT DOCUMENTS
`
`N
`
`i58
`1985,
`(Continued)
`
`OTHER PUBLICATIONS
`
`Berensmeier (Magnetic particles for the separation and purification
`of nucleic acids, Appl Microbiol Biotechnol (2006) 73:495-504).*
`(Continued)
`
`Primary Examiner — Stephanie K Mummert
`Assistant Examiner — Aaron Priest
`(74) Attorney, Agent, or Firin — Casimir Jones S.C.
`
`(57)
`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.
`
`16 Claims, 17 Drawing Sheets
`
`Geneoscopy Exhibit 1028, Page 1
`
`

`

`US 9,163.278 B2
`Page 2
`
`(56)
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`Microbiol., 2003, 41(11):5041-5045.
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`retrospective.” Biotechniques, 2008, 44:701-704.
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`
`Geneoscopy Exhibit 1028, Page 2
`
`

`

`US 9,163.278 B2
`Page 3
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`
`* cited by examiner
`
`Geneoscopy Exhibit 1028, Page 3
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 1 of 17
`
`US 9,163,278 B2
`
`FIG. 1A
`
`1. Prepare clarified supernatant
`Mix 8g stool with buffer
`Centrifuge
`Collect supernatant
`Treat With inhibitor removal resin
`Spin filter
`Recover clarified supernatant
`
`2. Sequential Capture of DNAs of Interest
`Add Guanidine isothiocyanate, heat
`Add oligonucleotide-conjugated paramagnetic beads
`(Alternatively, add the conjugated beads to the Guanidine
`Sothiocyanate Solution, then heat
`Hybridize
`
`3. Isolate Captured DNA
`Pull beads from Solution With madnet
`Remove supernatant
`(re-use supernatant for capture of next target, Fig. 1B)
`Wash Beads 3X
`Eute DNA
`
`Geneoscopy Exhibit 1028, Page 4
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 2 of 17
`
`US 9,163,278 B2
`
`FIG. 1B
`
`Prepare stool
`supernatant from
`homogenate
`
`Treat with PWPP to
`refore FCR inhibitors
`- clarified supernatant
`
`satata
`
`to
`
`
`
`
`
`
`
`
`
`t
`
`
`
`t
`
`ge
`
`
`
`Proceed with washing
`then elute DNA from
`beads; DNA is ready for
`(mock) bisulfite
`treater
`
`age to
`Place
`separate assister
`seats
`
`SE S
`
`age
`At
`estate at
`
`s
`
`Geneoscopy Exhibit 1028, Page 5
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 3 of 17
`
`US 9,163,278 B2
`
`FIG. 2
`
`
`
`Geneoscopy Exhibit 1028, Page 6
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 4 of 17
`
`US 9,163,278 B2
`
`FIG. 3
`
`
`
`Geneoscopy Exhibit 1028, Page 7
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 5 Of 17
`
`US 9,163,278 B2
`
`S
`FIG. 5A
`
`FIG. 5B
`
`65 2
`F.G. 5C
`
`Geneoscopy Exhibit 1028, Page 8
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 6 of 17
`
`US 9,163,278 B2
`
`FIG. 6
`
`
`
`Geneoscopy Exhibit 1028, Page 9
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 7 Of 17
`
`US 9,163,278 B2
`
`FIG. 7
`
`
`
`Geneoscopy Exhibit 1028, Page 10
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 8 of 17
`
`US 9,163,278 B2
`
`FIG. 8
`
`
`
`Geneoscopy Exhibit 1028, Page 11
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 9 Of 17
`
`US 9,163,278 B2
`
`FIG. 9
`
`
`
`Stool Supernatant #1; Gene A
`
`s
`
`&
`
`:
`
`7.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`.
`Cycle
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`Geneoscopy Exhibit 1028, Page 12
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 10 of 17
`
`US 9,163,278 B2
`
`FIG
`
`9
`
`
`
`
`
`nua aero paens aan ases aerod
`
`
`
`
`
`
`
`
`
`nad ad pºmenans aun aseg und
`
`Cycle
`
`Geneoscopy Exhibit 1028, Page 13
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 11 of 17
`
`US 9,163,278 B2
`
`FIG. 10
`
`A PVPP30-50pm particle; No Spin Filtration
`
`600
`
`t
`
`3.
`
`r
`
`7.
`
`3.
`
`*
`.
`
`8
`
`.
`
`.
`
`.
`
`. 2, 2, 2
`
`28 33 3, 38 .
`Cycle
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`B PVPP 100-130pm particle; No Spin Filtration
`900
`800
`
`
`
`i.
`t
`
`:
`
`e
`
`s
`
`S.
`
`400
`300
`
`100
`
`8 .
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`.
`Cycle
`
`.
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`
`.
`
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`
`.
`
`.
`
`.
`
`Geneoscopy Exhibit 1028, Page 14
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 12 of 17
`
`US 9,163,278 B2
`
`FIG. 10
`
`C PVPP 100-130pm particle; With Spin Filtration
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
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`Geneoscopy Exhibit 1028, Page 15
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 13 of 17
`
`US 9,163,278 B2
`
`FIG. 11
`
`Absorbance
`
`
`0.0
`100.0
`200.0
`300.0
`
`400.0
`
`Time (minutes)
`
`0.5500
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`0.5000 |
`0.4500
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`
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`
`200.0
`
`300.0
`
`400.0
`
`Time (minutes)
`
`Geneoscopy Exhibit 1028, Page 16
`
`Geneoscopy Exhibit 1028, Page 16
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 14 of 17
`
`US 9,163,278 B2
`
`FIG. 12
`
`Two sequential extractions, Gene V
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`
`Geneoscopy Exhibit 1028, Page 17
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 15 Of 17
`
`US 9,163,278 B2
`
`FIG. 13
`
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`
`Geneoscopy Exhibit 1028, Page 18
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 16 of 17
`
`US 9,163,278 B2
`
`C Gene V
`
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`
`Geneoscopy Exhibit 1028, Page 19
`
`

`

`U.S. Patent
`
`Oct. 20, 2015
`
`Sheet 17 Of 17
`
`US 9,163,278 B2
`
`F.G. 14
`
`Total
`Time(min)
`
`55
`
`Process B
`
`Prepare clarified supernatant
`Mix 3 g stool with 7 vols buffer
`Centrifuge
`Collect supernatant
`Filter through 0.45 um filter
`
`Total
`Time(min)
`
`minutes)
`
`15
`
`30
`
`Recover clarified supernatant
`Precipitate with isopropanol to
`remove intrinsic streptavidin
`Centrifuge; discard supernatant
`Dissolve pellet in 4.9 ml TE buffer
`H overnight-H
`Sequential Capture of DNAs of
`Parallel Capture of DNAs of
`interest
`interest
`Add Guanidine isothiocyanate to 10
`To a 300 uL aliquot, add guanidine
`ml, heat
`isothiocyanate and biotinylated
`probe oligonucleotide
`Hybridize
`
`Process A
`
`Prepare clarified supernatant
`Mix 8g stool with 32 m buffer
`Centrifuge
`Collect supernatant (24 ml)
`Treat 14 m with inhibitor removal
`resin
`Spin filter
`Recover clarified supernatant
`
`Add oligonucleotide-Conjugated
`paramagnetic beads
`Hybridize (60 minute per cycle)
`
`Add streptavidin-coated
`paramagnetic beads
`Hybridize oligonucleotide/DNA
`complexes to beads
`solate Captured DNA
`Pull beads from Solution with
`magnet
`Remove and discard fluid
`
`60
`
`30
`
`160
`
`isolate Captured DNA
`Pull beads from Solution with
`magnet
`Remove supernatant
`(reserve supe for next target
`capture)
`Wash Beads
`Wash Beads
`Eute DNA
`Eute DNA
`Total Time of Purification minutes
`Total Time of Purification minutes
`Equivalent Mass of Stool Yielding
`Equivalent Mass of Stool Yielding
`Purified Specific Gene DNA for
`Purified Specific Gene DNA for
`2.0 gram
`Amplification
`Amplification
`*See text for improved magnet configuration
`
`15
`
`15
`
`1020
`
`0.18 gram
`
`Geneoscopy Exhibit 1028, Page 20
`
`

`

`1.
`SOLATION OF NUCLECACDS
`
`US 9,163,278 B2
`
`The present application is a continuation of PCT/US2012/
`03751, filed May 11, 2012, which claims the benefit of U.S.
`Provisional Patent Application Ser. Nos. 61/485,214, 61/485,
`338, 61/485,386, and 61/485,448, each of which was filed
`May 12, 2011, and each of which is incorporated herein by
`reference in its entirety.
`
`FIELD OF INVENTION
`
`10
`
`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.
`
`15
`
`BACKGROUND
`
`Isolating specific target nucleic acids from a sample is an
`important step for many medical diagnostic assays. For
`example, certain mutations and methylation states in known
`genes are correlated, associated, and/or predictive of disease.
`DNA harboring these genes can be recovered from a sample
`and tested for the presence of the particular mutations and
`methylation states.
`In practice, such assays require isolating and assaying sev
`eral genetic targets from a sample. For many detection meth
`ods, detecting rare mutations or methylation events in a single
`gene requires isolating and testing a large quantity of DNA.
`This problem is compounded when assaying a panel of genes,
`each of which must be present in a large quantity for a robust
`diagnostic test. Thus, to detect rare mutations and methyla
`tion events in multiple genes, the isolated DNA must be
`highly concentrated and comprise a Substantial portion of the
`detection assay.
`This requirement imposes many problems, however. For
`example, preparing Such quantities and concentrations of
`DNA requires a large sample as input (e.g., having a mass of
`several grams, e.g., approximately 2-4 grams) to provide
`Sufficient nucleic acid for detection, and thus requires a
`method that can prepare DNA from a large sample. In addi
`tion, assay inhibitors are often isolated and concentrated with
`the DNA preparation. Consequently, concentrated DNA
`preparations produced by conventional methods also often
`retain unacceptable concentrations of inhibitors, which are
`then introduced into a Subsequent assay. Moreover, if all
`targets of the panel are extracted simultaneously in a bulk,
`non-selective DNA preparation, the sensitivity of the assay is
`compromised because, as the preparation is divided into ali
`quots for testing, less extracted DNA from any one gene of the
`panel is present in the assay. If, on the otherhand, all members
`of the panel are extracted and tested together and are thus
`present in the same assay mixture, the sensitivity of detecting
`any single particular target is compromised by the presence of
`the non-target DNA molecules.
`In addition, if a particular diagnostic target is present in a
`complex sample, it will be present in a small amount relative
`to other materials—both nucleic acid and non-nucleic acid—
`in the sample, thus providing a challenge for analytical meth
`ods designed to detect it. For example, analyses of DNA from
`stool samples is complicated by the fact that bacteria compose
`approximately 60% of the dry mass of feces and the remain
`der is largely the remains of plant and animal matter ingested
`as food by the Subject. As such, the human Subject’s cells,
`which are only those that slough off the lining of the digestive
`tract, are a very Small fraction of the stool and Substantial
`amounts of nucleic acids from other sources are present.
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`Furthermore, in assays to detect gene modifications indica
`tive of colon cancer, cells derived from a tumor that may be
`present in the colon would compose only a small fraction of
`the human Subject's gut cells that slough off the digestive tract
`lining. Consequently, cancer cells (and the DNAS they con
`tain) make up a minimal amount of the stool mass. Such
`samples are also often very viscous, which presents problems
`in sample preparation and isolation of nucleic acid.
`Conventional methods and kits for isolating DNA from
`samples typically prepare total DNA (e.g., by a non-specific
`precipitation method) from a sample. For complex samples
`Such as stool samples, this is a particular drawback of con
`ventional methods, as total DNA isolated from a stool sample
`comprises DNA from the gut-resident bacteria (and any
`viruses, eukaryotes, and archaea present) along with DNA
`from the subject. Moreover, conventional methods and kits
`are primarily designed to prepare DNA from Small samples,
`e.g., samples having masses of less than 1 gram, e.g., 50 to
`200 milligrams, limiting the yield of target nucleic acid from
`complex samples to very Small amounts. Additional draw
`backs are that most conventional technology does not effec
`tively remove inhibitors and often require long processing
`steps, e.g., incubations. Consequently, conventional methods
`are not Suited to high-sensitivity and high-specificity multi
`gene panel analysis because they cannot prepare sufficient
`amounts of highly concentrated, inhibitor-free DNA from
`large samples, such as a stool sample of several grams. Assays
`using DNA prepared with conventional methods will not
`provide a sample that can be assayed with the required sen
`sitivity threshold for detecting rare mutation or methylation
`events. Using a conventional method or kit to attain the start
`ing quantities needed to attain such sensitivity requires mul
`tiple DNA extractions (e.g., the use of multiple kits) from
`multiple samples in addition to extra purification steps to
`remove inhibitors. Therefore, what is needed is a method of
`preparing concentrated, inhibitor-free DNA from a sample
`for each member of a gene panel for use in diagnostic assays.
`
`SUMMARY
`
`Provided herein is technology relating to isolating nucleic
`acids. In particular, the technology relates to methods, sys
`tems, and kits for extracting and purifying nucleic acids from
`exfoliated intestinal cells in stool specimens for use in quan
`titative and sensitive assays. The technology is embodied in a
`novel method for purifying specific DNA from stool that
`utilizes inhibitor removal steps and direct capture of DNA
`from stool Supernatant, or a combination of these steps. The
`technology further provides filtration devices suitable for use
`with complex and Viscous samples, such as stool samples.
`Accordingly, provided herein is a method for isolating a target
`nucleic acid from a sample, the method comprising removing
`an assay inhibitor, if present, from the sample to produce a
`clarified sample; capturing the target nucleic acid, if present,
`from the clarified sample with a capture reagent to form a
`capture complex; isolating the capture complex from the
`clarified sample; and recovering the target nucleic acid, if
`present, from the capture complex in a nucleic acid solution.
`In some embodiments the method further comprises retaining
`the clarified sample after the capturing step; and repeating the
`isolating and recovering steps using the retained clarified
`sample and a second capture reagent.
`In some embodiments, removing the inhibitor comprises
`homogenizing the sample to produce a homogenate; centri
`fuging the homogenate to produce a Supernatant; treating the
`Supernatant with an inhibitor-adsorbing composition to bind
`the inhibitor, if present, in an inhibitor complex; and isolating
`
`Geneoscopy Exhibit 1028, Page 21
`
`

`

`3
`the inhibitor complex from the Supernatant to produce a clari
`fied sample. The inhibitor-adsorbing composition in some
`embodiments is a polyvinylpyrrolidone. In some embodi
`ments, the polyvinylpyrrolidone is insoluble and in some
`embodiments the polyvinylpyrrolidone is a polyvinylpoly
`pyrrolidone. It is useful in some embodiments to provide the
`polyvinylpyrrolidone in a premeasured form, for example in
`Some embodiments the polyvinylpyrrolidone is provided as a
`tablet. Various techniques are used to separate the inhibitor
`complex from the sample. For example, in some embodi
`ments isolating the inhibitor complex comprises centrifuging
`to separate the inhibitor complex from the Supernatant.
`In some embodiments, the centrifuging comprises centri
`fuging through a spin column. Therefore, in Some embodi
`ments provided herein is technology relating to filtration and
`particularly, but not exclusively, to filters and methods for
`filtering by means of centrifugation. Specifically, some
`embodiments of the technology provided herein address the
`problem of spin filter clogging by providing technology in
`which both the bottom end and body of a spin filter are made
`from a porous or permeable material. That is, the walls of the
`spinfilterare made of the same or similar material as that used
`for the filter means at the bottom end in conventional designs.
`As such, when the bottom portion of the filter becomes
`clogged during filtration, the walls provide additional Surface
`through which the sample can be filtered.
`This technology is provided hereinas a spin filter compris
`ing a hollow body, a bottom end, and an open top end opposite
`the bottom end, wherein the hollow body is made from a
`porous filtering material. In some embodiments the bottom
`end is made from a porous filtering material. The hollow body
`and bottom end of the spin filter assume any shape appropri
`ate for the filtration application to which the filter is applied.
`For example, in some embodiments the hollow body is a tube
`and in some embodiments the bottom end is a hemisphere. In
`other embodiments, the bottom end is a disc, a cone, or a
`portion of an ellipsoid. Furthermore, the spin filter is made
`from any material that is appropriate for filtering a sample.
`Thus, in some embodiments the porous filtering material is
`polyethylene. Samples comprise varying sizes of particles,
`matter, precipitates, etc. that are to be removed by filtration.
`Accordingly, the filtering material can be selected to have
`physical properties that provide the desired separation. For
`example, in Some embodiments the porous filtering material
`has a nominal pore size of 20 micrometers. In some embodi
`ments, use of the filterproduces a filtrate that a user retains for
`additional processing. As such, Some embodiments provide a
`spin filter assembly comprising a spin filter as described and
`a collection vessel adapted to receive the spinfilter and collect
`the filtrate.
`Also provided herein are methods for producing a filtrate
`from a sample comprising placing a sample to be filtered into
`the spin filter and centrifuging the spin filter, wherein during
`centrifuging, a fraction of the sample passes through porous
`filtering material of said spin filter to produce a filtrate.
`The technology can be provided as a kit for use in a sample
`separation. Embodiments of such a kit comprise a spin filter
`as described and an instruction for use. In some embodiments
`the kit further comprises a collection vessel. In some embodi
`ments, a kit comprising a spin filter further comprises addi
`tional reagents and materials for sample preparation, e.g., for
`inhibitor removal and/or target nucleic acid isolation.
`In some embodiments, the methods and sy