An apparatus for nucleic acid preparation from cells comprising housings, chambers, piercing elements, valves and pumps

AN APPARATUS FOR NUCLEIC ACID PREPARATION FROM CELLS COMPRISING HOUSINGS, CHAMBERS, PIERCING ELEMENTS, VALVES AND PUMPS [0001]

BACKGROUND OF THE INVENTION

·) Field of the Invention [0002]

The present invention relates to the field of biotechnology devices and, in particular, to devices and methods for preparing samples.

2.) Discussion of Related Art [0003] DNA can be used to develop new drugs or to link someone to a crime. However, before this can be done, the DNA must be isolated from a sample. These samples include, for example, blood, urine, human cells, hair, bacteria, yeast and tissue. Each of these samples include cells, which include nucleic acid. Nucleic acid is a nucleotide chain, which conveys genetic information. The most common forms of nucleic acid are DNA and RNA.

[0004] In order to isolate the nucleic acid from the samples, prior art devices use a tray having several exposed cavities. The sample is placed into one of the cavities and conventional processing steps are used to isolate the DNA from the sample.

CA 2912883 2018-05-04

CA 02912883 2015-11-19 [00051 This prior art system has several disadvantages, including contamination. Since the cavities are exposed, contaminants can easily affect the DNA. In addition, the prior art system requires the preparation of several samples at one time. It is difficult to prepare one or two samples at a time using the prior art devices.

SUMMARY OF THE INVENTION [0006] A cassette for preparing a sample is disclosed herein. The cassette includes at least one mixing chamber for receiving a sample of cells; a first holding chamber; an enzyme in the first holding chamber, the enzyme being transferable into the at least one mixing chamber to break the cells and release nucleic acid from the cells to create bulk material and the nucleic acid in the bulk material; a second holding chamber; magnetic particles in the second holding chamber, the magnetic particles being transferable to the at least one mixing chamber to bind with the nucleic acid; and at least one magnet, positionable to attract the magnetic particles together with the nucleic acid and at least partially separate the nucleic add from the bulk material in the at least one mixing chamber.

[0007] The mixing chamber has a top surface, the top surface having an opening therein. A removable lid for accessing the opening of the mixing chamber is also provided. The enzyme may be proteinase K. First and second plungers in the first holding chamber and second holding chamber may also be provided, each plunger being movable to transfer the enzyme and magnetic particles, respectively, into the mixing chamber. A thin film that is breakable to transfer the enzyme and magnetic particles through respective ruptures in the thin film into the mixing chamber may also be provided. First and second plungers in the first holding chamber and second holding chamber, each plunger being movable to break the thin film and transfer the enzyme and magnetic partides, respectively into the mixing

CA 02912883 2015-11-19 chamber, may also be provided. A third holding chamber and a lysis solution in the third holding chamber may also be provided, the lysis solution being transferable into the mixing chamber to solubilize the bulk material. A fourth holding chamber and a binding solution in the fourth holding chamber may also be provided, the binding solution being transferable into the mixing chamber to bind the nucleic acid to the magnetic particles. A heating element for heating the mixing chamber may also be provided.

[00081 The cassette may also include a first separation piece having a surface; a first transfer piece having a surface with a cavity therein, the at least one magnet transferring the magnetic particles together with the nucleic acid into the cavity in the surface of the first transfer piece, the first transfer piece being movable relative to the first separation piece so that the magnetic particles together with the nucleic acid move out of the mixing chamber and past the surface of the first separation piece; and a first receiving chamber which receives the magnetic particles and nucleic acid after moving past the surface of the first separation piece. The cassette may further include a second separation piece having a surface; a second transfer piece having a surface with a cavity therein, the second transfer piece being movable relative to the second separation piece so that the magnetic particles together with the nucleic acid move out of the first receiving chamber and past the surface of the second separation piece; and a second receiving chamber which receives the magnetic particles and nucleic add after moving past the surface of the second separation piece. The cassette may yet further include a third separation piece having a surface; a third transfer piece having a surface with a cavity therein, the third transfer piece being movable relative to the third separation piece so that the magnetic particles together with the nucleic acid move out of the second receiving chamber and past the surface of the third separation piece; and a third

CA 02912883 2015-11-19

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receiving chamber which receives the magnetic particles and nucleic add [0009] The first receiving chamber may be a washing chamber, and the cassette may further include a washing solution in the washing chamber. The second receiving chamber may be a washing chamber, and the cassette may further include a washing solution in the washing chamber. The third receiving chamber may be an elution chamber, and the cassette may further include an elution buffer in the elution chamber for separating the magnetic particles and the nucleic acid.

[0010] A cassette for preparing a sample is disclosed herein. The cassette includes at least one mixing chamber for receiving a sample of cells, an enzyme being added in the mixing chamber to break the cells and release nucleic add from the cells to create bulk material and nucleic add in the bulk material, magnetic particles being added to the mixing chamber to bind with the nucleic add; a first separation piece having a surface; a first transfer piece having a surface with a cavity therein; a magnet, positionable to attract the magnetic particles together with the nudeic add and at least partially separate the nucleic add from the bulk material in the at least one mixing chamber and to transfer the magnetic partides together with the nucleic acid into the cavity in the surface of the first transfer piece, the first transfer piece being movable relative to the first separation piece so that the magnetic partides together with the nucleic acid move out of the mixing chamber and past the surface of the first separation piece; and a first receiving chamber which receives the magnetic particles and nucleic add after moving past the surface of the first separation piece.

[0011] The second piece may be rotatable relative to the first piece. The first receiving chamber may be a washing chamber, and the cassette may further indude a washing solution in the washing chamber. The cassette may also indude an elution chamber, and an elution buffer in the elution

CA 02912883 2015-11-19 chamber for separating the magnetic particles and the nucleic acid. The cassette may also include a second separation piece having a surface, a second transfer piece having a surface with a cavity therein, the second transfer piece being movable relative to the second separation piece so that the magnetic particles together with the nucleic acid move out of the first receiving chamber and past the surface of tire second separation piece, the elution chamber receiving the magnetic particles and nucleic acid after moving past the surface of the first separation piece. The second transfer piece may be moveable relative to the second separation piece so that the magnetic particles move out of the elution chamber, leaving the nucleic acid in the elution chamber.

[0012] The secondary chamber may be an elution chamber and the cassette may further include an elution buffer in the elution chamber for separating the magnetic particles and the nucleic acid. The cassette may also include a second separation piece having a surface, a second transfer piece having a surface with a cavity therein, the second transfer piece being movable relative to the second separation piece so that the magnetic particles together with the nucleic acid move out of the first receiving chamber and past the surface of the second separation piece, and a second receiving chamber which receives the magnetic particles and nucleic add after moving past the surface of the second separation piece. The cassette may further include a third separation piece having a surface, a third transfer piece having a surface with a cavity therein, the third transfer piece being movable relative to the third separation piece so that the magnetic particles together with the nucleic add move out of the second receiving chamber and past the surface of the third separation piece, and a third receiving chamber which receives the magnetic partides and nucleic add after moving past the surface of the third separation piece. The second receiving chamber may be a washing chamber, and the cassette may further indude a

CA 02912883 2015-11-19 washing solution in the washing chamber. The third receiving chamber may be an elution chamber, and the cassette may further include an elution buffer in the elution chamber for separating the magnetic particles and the nucleic add.

[0013] A cassette for preparing samples is disclosed herein. The cassette includes an enclosure, the enclosure comprising a mixing chamber, the mixing chamber including an opening for receiving a sample of cells having nucleic add; a plurality of holding chambers having contents, the contents of one of the plurality of holding chambers comprising magnetic partides, and the contents of one of the plurality of holding chambers comprising a proteinase K solution; a plurality of plungers, each of the plurality of plungers corresponding to one of the plurality of holding chambers, for transferring the contents of the plurality of holding chambers into the mixing chamber, the proteinase K solution breaking up the cells to release the nudeic add and the nucleic add binding to the magnetic particles in the mixing chamber; a first valve, coupled to the mixing chamber, the first valve including a positionable magnet for attracting the magnetic partides; a washing chamber, coupled to the first valve; a second valve, coupled to the first washing chamber, the second valve including a positionable magnet for attracting the magnetic particles; and an elution chamber, coupled to the second valve, the elution chamber induding an opening for removing the nudeic add from the elution chamber.

[0014] One of the plurality of holding chambers may include a bindiiig solution and one of the plurality of holding chambers may include a lysis solution, and wherein one of the plurality of plungers may transfer the binding solution into the mixing chamber and wherein one of the plurality of plungers may transfer the lysis solution into the mixing chamber.

[0015] A cassette for preparing a sample is disclosed herein. The cassette indudes a reaction chamber for receiving a sample of cells; a first holding

CA 02912883 2015-11-19 chamber; an enzyme in the first holding chamber, the enzyme being transferable into the reaction chamber to break the cells and release nucleic acid from the cells to create bulk material and the nucleic acid in the bulk material; a particle chamber; particles to bind with the nucleic acid in the particle chamber; a second holding chamber; an elution buffer in the second holding chamber to release the nucleic acid from the particles; and an elution chamber for receiving the elution buffer and the released nucleic add, wherein the elution buffer is transferable from the holding chamber to the reaction chamber, through the particle chamber, and into the elution chamber.

[0016] The cassette may include a third holding chamber and a lysis solution in the third holding chamber, the lysis solution being transferable into the reaction chamber to solubilize the bulk material.

[0017] The cassette may include a fourth holding chamber and a binding solution in the fourth holding chamber, the binding solution being transferable into the reaction chamber to bind the nucleic acid to the particles.

[0018] The reaction chamber may be aligned with the particle chamber and the elution chamber may be alignable with the particle chamber.

[0019] The cassette may include a waste chamber for receiving the enzyme and bulk material.

[0020] The cassette may include a plunger to transfer the contents of the reaction chamber through the partide chamber and into the waste chamber. [0021] The cassette may include a plunger to transfer the contents of the reaction chamber through the particle chamber and into the elution chamber.

[0022] The cassette may indude a valve in each of the first and second holding chambers to transfer the contents of each of the first and second holding chambers into the reaction chamber.

CA 02912883 2015-11-19 [0023] The valve may indude a plunger to transfer the contents from the valve into the reaction chamber.

10024] Another cassette for preparing samples is also disclosed herein. The cassette indudes an enclosure, the endosure induding a reaction chamber, the reaction chamber induding an opening for receiving a sample of cells having nucleic add; a plurality of holding chambers; an enzyme in one of the plurality of holding chambers; a lysis buffer in one of the plurality of holding chambers; a binding buffer in one of the plurality of holding chambers; an elution buffer in one of the plurality of holding chambers; a partide chamber; particles in the partide chamber, the partides to releasably bind with the nucleic add; and an elution chamber to receive the released nucleic add, the elution chamber including an opening for removing the nucleic acid from the enclosure.

[0025] The endosure may indude a waste chamber.

[0026] Each of the plurality of holding chambers may indude a plunger for transferring contents of the plurality of holding chambers to the reaction chamber.

[0027] The cassette may include one or more washing buffers in one or more of the plurality of holding chambers.

[0028] The reaction chamber may be aligned with the particle chamber and wherein the elution chamber may be alignable with the partide chamber. [0029] A further cassette for preparing a sample is also disdosed herein. The cassette indudes a reaction chamber for receiving a sample of cells; a first holding chamber; an enzyme in the first holding chamber, the enzyme being transferable into the reaction chamber to break the cells and release nudeic add from the cells to create bulk material and the nucleic add in the bulk material; a partide chamber; particles to bind with the nudeic add in the particle chamber; a second holding chamber; an elution buffer in the second holding chamber to release the nudeic add from the partides; and

CA 02912883 2015-11-19 an elution chamber for receiving the elution buffer and the released nucleic add, wherein each of the holding chambers comprises an outer housing having a first chamber therein and at least one opening, the first chamber receiving a valve, the valve comprising: an inner housing having a second diamber therein and at least one opening, the inner housing rotatable relative to the outer housing, the at least one opening of the inner houeing alignable with the at least one oper.ir.g of the outer housing, the second chamber having contents; and a plunger in the second chamber to transfer the contents of the second chamber through the at least one opening of the inner housing and the at least one opening of the outer housing when the at least one opening of the inner housing and the at least one opening of the outer housing are aligned.

[0030] Hie cassette may include a third holding chamber and a lysis solution in the third holding chamber, the lysis solution being transferable into the reaction chamber to solubilize the bulk material.

[0031] The cassette may indude a fourth holding chamber and a binding solution in the fourth holding chamber, the binding solution being transferable into the reaction chamber to bind the nucleic acid to the par tides.

[0032] The cassette may indude a waste chamber for receiving the enzyme and bulk material.

[0033] Tlie cassette may include a plunger to transfer the contents of the reaction chamber through the particle chamber and into the waste chamber. [0034] The cassette may include a plunger to transfer the contents of the reaction chamber through the particle chamber and into the elution chamber.

[0035] The reaction chamber may be aligned with the partide chamber and wherein the elution chamber may be alignable with the particle chamber.

CA 02912883 2015-11-19

In a further aspect, the present disclosure provides a method, including moving a plunger within a reagent chamber of a cartridge such that the plunger punctures a portion of a puncturable member defining a boundary of the reagent chamber, the moving the plunger including transferring a plurality of magnetic particles from the reagent chamber to a first chamber defined by a housing of the cartridge, disposing a magnetic shaft at least partially within a movable valve member of a valve to attract the plurality of magnetic particles into a cavity defined by the movable valve member, the valve at least partially disposed within the housing, the valve including the movable valve member and a valve housing, and rotating the movable valve member relative to the valve housing to transfer the plurality of magnetic particles between the first chamber of the housing and a second chamber defined by the housing while sealing the plurality of magnetic particles within the cavity.

In an embodiment disclosed, the method further includes removing the magnetic shaft from within the movable valve member of the valve to release the plurality of magnetic particles from the cavity.

In an embodiment disclosed, the valve is a first valve, the method further including disposing the magnetic shaft at least partially within a movable valve member of a second valve to attract the plurality of magnetic particles into a cavity defined by the movable valve member of the second valve, the second valve at least partially disposed within the housing, the second valve including the movable valve member and a valve housing, and moving the valve member of the second valve relative to the valve housing of the second valve to transfer the plurality of magnetic particles between the second chamber of the housing and a third chamber defined by the housing.

In an embodiment disclosed, the method further includes removing the magnetic shaft from within the movable valve member of the second valve to release the plurality of magnetic particles from the cavity defined by the movable valve member of the second valve.

9a

CA 02912883 2015-11-19

In an embodiment disclosed, the valve member is moved relative to the valve housing by rotating the magnetic shaft disposed at least partially within the valve member.

In an embodiment disclosed, the puncturable member is a thin film.

In a further aspect, the present disclosure provides a method for isolating nucleic adds from a sample including receiving a sample in a mixing chamber of a cassette, the cassette comprising the mixing chamber, a first washing chamber, a first valve comprising a valve housing defining a first valve opening in fluid communication with the mixing chamber and a second valve opening in fluid communication with the first washing chamber, a first reagent chamber configured to contain a lysis buffer, a second reagent chamber configured to contain magnetic particles, and a third reagent chamber configured to contain a binding buffer, wherein the first, second, and third reagent chambers each include a puncturable portion defining at least a portion of a boundary between the mixing chamber and the first, second, and third reagent chambers, transferring the lysis buffer to the mixing chamber by puncturing the puncturable portion of the first reagent chamber, transferring the magnetic particles to the mixing chamber by puncturing the puncturable portion of the second reagent chamber, transferring the binding buffer to the mixing chamber by puncturing the puncturable portion of the third reagent chamber, binding nucleic acids in the sample to the magnetic particles, receiving the magnetic particles and the bound nucleic acids in a cavity defined by a valve member disposed in the valve housing, and transferring the magnetic particles and the bound nucleic acids from the mixing chamber to the first washing chamber by moving the valve member relative to the valve housing from a first position in which the cavity is in fluid communication with the mixing chamber to a second position in which the cavity is in fluid communication with the washing chamber.

In an embodiment disclosed, the puncturable portion of the first reagent chamber is punctured by a piercing element of a plunger in the first reagent chamber.

9b

CA 02912883 2015-11-19

In an embodiment disclosed, the puncturable portion of the second reagent chamber is punctured by a piercing element of a plunger in the second reagent chamber.

In an embodiment disclosed, the puncturable portion of the third reagent chamber is punctured by a piercing element of a plunger in the third reagent chamber.

In an embodiment disclosed, the cassette further comprises a fourth reagent chamber configured to contain proteinase K (PK), wherein the fourth chamber includes a puncturable portion defining at least a portion of a boundary between the mixing chamber and the fourth reagent chamber.

In an embodiment disclosed, the method further includes transferring the PK to the mixing chamber by puncturing the puncturable portion of the fourth reagent chamber.

In an embodiment disclosed, the PK is transferred to the mixing chamber prior to adding the lysis buffer to the mixing chamber.

In an embodiment disclosed, the cassette further comprises a pump in fluid communication with the mixing chamber.

In an embodiment disclosed, the pump is a bellows pump.

In an embodiment disclosed, the method further includes actuating the pump to mix the sample with one or more of the lysis buffer, magnetic particles, or binding buffer in the mixing chamber.

In an embodiment disclosed, the receiving and the transferring of the magnetic particles comprises inserting a magnet into the valve member to attract the magnetic particles and the bound nucleic acids to the cavity when the valve is in the first position, moving the valve to the second position, and withdrawing the magnet from the valve member.

9c

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In an embodiment disclosed, the method further includes heating the sample and the lysis buffer in the mixing chamber.

In an embodiment disclosed, the method further includes transferring a first washing solution to the first washing chamber, and mixing the washing solution with the magnetic particles and the bound nucleic acids in the first washing chamber.

In an embodiment disclosed, the cassette further includes a second valve comprising a valve housing defining a first valve opening in fluid communication with the first washing chamber and a second valve opening in fluid communication with a second washing chamber, and a third valve comprising a valve housing defining a first valve opening in fluid communication with the second washing chamber and a second valve opening in fluid communication with an elution chamber.

In an embodiment disclosed, the method further includes receiving the magnetic particles and the bound nucleic acids in a cavity defined by a valve member disposed in the valve housing of the second valve, transferring the magnetic particles and the bound nucleic acids from the first washing chamber to the second washing chamber by moving the valve member relative to the valve housing of the second valve from a first position in which the cavity is in fluid communication with the first washing chamber to a second position in which the cavity is in fluid communication with the second washing chamber, transferring a second washing solution to the second washing chamber, and mixing the washing solution with the magnetic particles and the bound nucleic acids in the second washing chamber, receiving the magnetic particles and the bound nucleic acids in a cavity defined by a valve member disposed in the valve housing of the third valve, and transferring the magnetic particles and the bound nucleic acids from the second washing chamber to the elution chamber by moving the valve member relative to the valve housing of the third valve from a first position in which the cavity is in fluid communication with the second washing chamber to a second position in which the cavity is in fluid communication with the elution chamber.

9d

CA 02912883 2015-11-19

In an embodiment disclosed, the method further includes eluting the nucleic acids from the magnetic particles by transferring elution buffer to the elution chamber, and mixing the elution buffer with the magnetic particles and the nucleic acids in the elution chamber.

In an embodiment disclosed, the method further includes heating the elution chamber.

In an embodiment disclosed, the method further includes receiving the magnetic particles in the cavity defined by the valve member disposed in the valve housing of the third valve, and transferring the magnetic particles from the elution chamber to the second washing chamber by moving the valve member relative to the valve housing of the third valve from the second position in which the cavity is in fluid communication with the elution chamber to the first position in which the cavity is in fluid communication with the second washing chamber.

In an embodiment disclosed, the puncturable portion of the first, second, and third reagent chambers is a thin film.

In an embodiment disclosed, the sample comprises cells.

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BRIEF DESCRIPTION OF THE DRAWINGS [0036] The invention is described by way of example with reference to the accompanying drawings, wherein:

[0037] FIG. 1 is a cross-sectional side view of a cassette for preparing samples according to an embodiment of the invention;

[0038] FIG. 2 is a perspective view of a cassette for preparing samples according to an embodiment of the invention;

[0039] FIG. 3 is a cross-sectional side view showing a sample being placed into the cassette using a pipette, according to an embodiment of the invention;

[0040] FIG. 4 is a perspective view of a magazine, in which the cassette of FIG. 1 is used, according to an embodiment of the invention;

[0041] FIG. 5 is a perspective view of an instrument, in which the magazine of FIG. 4 is used, according to an embodiment of the invention;

[0042] FIG. 6 is a cross-sectional side view of the cassette of FIG. 1, showing the transfer of a PK solution into a mixing chamber, according to an embodiment of the invention;

[0043] FIG. 7 is a cross-sectional side view of the cassette of FIG. 1, showing the transfer of a lysis solution into a mixing chamber, according to an embodiment of the invention;

[0044] FIG. 8 is a cross-sectional side view of the cassette of FIG. 1, showing the transfer of a binding solution into a mixing chamber, according to an embodiment of the invention;

[0045] FIG. 9 is a cross-sectional side view of the cassette of FIG. 1, showing the transfer of metallic beads into the mixing chamber, according to an embodiment of the invention;

CA 02912883 2015-11-19 [0046] FIG. 10 is a cross-sectional side view of the cassette of FIG. 1, showing metallic beads bound to a first valve, according to an embodiment of the invention;

[0047] FIG. 11 is a cross-sectional side view of the cassette of FIG. 1, showing tlie transfer of metallic beads from a mixing chamber to a washing chamber, according to an embodiment of the invention;

[0048] FIG. 12 is a perspective end view of a valve for use in the cassette of FIG. 1 according to an embodiment of the invention;

[0049] FIG. 13 is a cross-sectional side view of the cassette of FIG. 1, showing metallic beads bound to a second valve, according to an embodiment of the invention;

[0050] FIG. 14 is a cross-sectional side view of the cassette of FIG. 1, showing the transfer of metallic beads from a first washing chamber to a second washing chamber, according to an embodiment of the invention;

[0051] FIG. 15 is a cross-sectional side view of the cassette of FIG. 1, showing metallic beads bound to a third valve, according to an embodiment of the invention;

[0052] FIG. 16 is a cross-sectional side view of the cassette of FIG. 1, showing the transfer of metallic beads from a second washing chamber to an elution chamber, according to an embodiment of the invention;

[0053] FIG. 17 is a cross-sectional side view of the cassette of FIG. 1, showing transfer of metallic beads from an elution chamber to a second washing chamber, according to an embodiment of the invention;

[0054] FIG. 18 is a cross-sectional side view of the cassette of FIG. 1, showing removal of a prepared sample from an elution chamber, according to an embodiment of the invention;

[0055] FIG. 19 is a perspective view of a magazine in which a multichannel pipette is used to access a plurality of samples from a plurality of cassettes;

CA 02912883 2015-11-19 [0056] FIG. 20 is a cross-sectional perspective view of an alternative embodiment of the cassette of FIG. 1 according to an embodiment of the invention;

[0057] FIG. 21 is a detailed perspective view of an assembly component of the cassette of FIG. 20 according to an embodiment of the invention;

[0058] FIG. 22 is a detailed perspective view of a plunger of the cassette of FIG. 20 according to an embodiment of the invention;

[0059] FIG. 23 is a detailed perspective view of a valve of the cassette according to an embodiment of the invention; and [0060] FIG. 24 is a detailed perspective view of the valve of FIG. 23 according to an embodiment of the invention.

[0061] FIG. 25 is a perspective view of a cassette for preparing samples according to one embodiment of the invention;

[0062] FIG. 26 is a partial cross-sectional view of the cassette of FIG. 25; [0063] FIG. 27 is a partial cross-sectional perspective view of a valve for use in the cassette of FIG. 25;

[0064] FIG. 28 is a partial cross-sectional side view of the valve of FIG. 25; [0065] FIG. 29 is a cross-sectional perspective view of the cassette of FIG.

25, showing the addition of a sample into a mixing chamber, according to an embodiment of the invention;

[0066] FIG. 30 is a .cross-sectional perspective view of the cassette of FIG. 25, showing the transfer of a PK solution into a mixing chamber, according to an embodiment of the invention;

[0067] FIG. 31 is a cross-sectional perspective view of the cassette of FIG. 25, showing the transfer of a lysis solution into a mixing chamber, according to an embodiment of the invention;

[0068] FIG. 32 is a cross-sectional perspective view of the cassette of FIG. 25, showing the transfer of a binding solution into a mixing chamber, according to an embodiment of the invention;

CA 02912883 2015-11-19 [0069] FIG. 33 is a cross-sectional perspective view of the cassette of FIG.

25, showing transfer of a sample through particles and into a waste chamber, according to an embodiment of the invention;

[0070] FIG. 34 is a cross-sectional perspective view of the cassette of FIG. 25, showing the pumping of a wash buffer into the mixing chamber, according to an embodiment of the invention;

[0071] FIG. 35 is a cross-sectional perspective view of the cassette of FIG. 25, showing the pumping of the wash buffer through the particles, according to an embodiment of the invention;

[0072] FIG. 36 is a cross-sectional perspective view of the cassette of FIG. 25, showing the pumping of a second wash buffer into the mixing chamber, according to an embodiment of the invention;

[0073] FIG. 37 is a cross-sectional perspective view of the cassette of HG. 25, showing the pumping of the second wash buffer through the particles, according to an embodiment of the invention;

[0074] FIG. 38 is a cross-sectional perspective view of the cassette of FIG. 25, showing the pumping of an elution buffer into the mixing chamber, according to an embodiment of the invention;

[0075] FIG. 39 is a cross-sectional perspective view of the cassette of FIG. 25, showing the pumping of the elution buffer through the particles and into the elution chamber, according to an embodiment of the invention; and [0076] FIG. 40 is a cross-sectional perspective view of the cassette of FIG.

25, showing the removal of the sample from the elution chamber, according to an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION [0077] Figure 1 illustrates a cassette 10, which can be used to prepare cell samples. The cassette 10 includes a housing 12, a mixing chamber 14, first, second third and fourth holding chambers 16,18,20 and 22, first, second,

CA 02912883 2015-11-19 third and fourth plungers 24,26,28 and 30, first, second and third valves 32, 34 and 36, first and second washing chambers 38 and 40, an elution chamber 42, first, second, third and fourth pumps 44,46,48 and 50, first and second lids 52 and 54, first and second heating elements 56 and 58 and a magnet 60. [0078] Each of the chambers 14,16,18,20,22,38,40 and 42, plungers 24, 26,28 and 30, valves 32,34 and 36, pumps 44,46,48 and 50, and heating elements 56 and 58 are enclosed within the housing. The lids 52 and 54 are movably attached to the housing 12. The magnet 60 is removably positionable in the first valve 32, second valve 34 and third valve 36.

[0079] The mixing chamber 14 has a top surface 62, a bottom surface 64 and opposing side surfaces 66,68. The top surface 62 of the mixing chamber includes an opening 70 therein.

[0080] The first lid 52 is configured to provide access to the opening 70 in the top surface 62 of the mixing chamber. The first lid 52 and the opening 70 are coaxial. The first lid 52 is shown being movably attached to the housing 12, such that when the lid 52 is open or off, the opening 70 is accessible and if the lid 52 is closed or on, the opening 70 is not accessible.

[0081] A thin film 74 forms one wall of the mixing chamber 14. The thin film 74 is breakable, such that the mixing chamber 14 is accessible when the thin film 74 has been broken or ruptured.

[0082] The first holding chamber 16, second holding chamber 18, third holding chamber 20 and fourth holding chamber 22 are shown located next to the mixing chamber 14 and aligned vertically with one another. Each of the holding chambers 16,18,20, 22 has an opening 76 next to the thin film 74 of the mixing chamber 14.

[0083] The cassette 10 further includes magnetic iron particles in the form of magnetic iron beads in the first holding chamber 16. The cassette 10 further includes a binding solution in the second holding chamber 18. The cassette 10 further includes a lysis solution in the third holding chamber 20.

CA 02912883 2015-11-19

The cassette 10 further includes a proteinase K (PK) solution in the fourth holding chamber 22.

[0084] The first, second, third and fourth plungers 24,26,28 and 30 are located in the first, second, third and fourth holding chambers 16,18,20 and 22, respectively.

[0085] Each of the plungers 16,18,20,22 includes a base 78, a shaft 80 and a piercing element 82. The shaft 80 extends from the base 78. The piercing element 82 is at the end of the shaft 80 opposing the base 78 and is pointed. The piercing element 82 is configured to break or rupture the thin film 74 of the mixing chamber 14.

[0086] The first pump 44 is a bellows pump having a pumping portion and a nozzle portion. The nozzle portion of the first pump 44 is located inside the mixing chamber 14. The pumping portion of the first pump 44 is located outside the mixing chamber, such that the pumping portion is actuatable. [0087] A heating element 56 is provided at the bottom surface 64 of the mixing chamber 14 for heating the contents of the mixing chamber 14. The heating element 56 may be a variable heating element.

[0088] The opposing side surface 68 of the mixing chamber 14 also includes an opening 84. A first valve 32 is provided between the opening 84 in the side 68 of the mixing chamber 14 and the first washing chamber 38.

[0089] The first valve 32 has a first stationary piece 86 and a second moveable piece 88, the second piece 88 being moveable relative to the first piece 86. The first stationary piece 86 includes a first opening 90 and a second opening 92 and has a surface 94. Ilie second piece 88 has an opening 94 therein for receiving the magnet 60. The second piece 88 has a surface 96 with a cavity 98 therein. The magnet 60 is shaped to correspond to the opening 94 in the second piece 88. The magnet 60 is moveable in the opening 94 of the second piece 88, and is removable from the second piece 88.

CA 02912883 2015-11-19 [0090] The cassette 10 includes a washing solution in the first washing chamber 38. The second pump 46 is also a bellows pump, and the nozzle portion of the second pump 46 is located in the first washing chamber 38. [0091] The second valve 34 is provided between the first washing chamber 38 and the second washing chamber 40. The second valve 34 is structurally and functionally the same as the first valve 43, and also includes a first stationary piece 86 and a second moveable piece 88. The first stationary piece 86 includes a first opening 90 and a second opening 92 and has a surface 94. The second moveable piece 88 has a surface 96 with a cavity 98 therein.

[0092] The cassette 10 includes a washing solution in the second washing chamber 40. The third pump 48 is also a bellows pump, and the nozzle portion of the third pump 48 is located in the second washing chamber 40. [0093] The third valve 36 is provided between the second washing chamber 40 and the elution chamber 42. The third valve 36 is structurally and functionally the same as the first valve 32 and the second valve 34, and also includes a first stationary piece 86 and a second moveable piece 88. The first stationary piece 86 includes a first opening 90 and a second opening 92 and has a surface 94. The second moveable piece 88 has a surface 96 with a cavity 98 therein.

[0094] The cassette 10 includes a washing solution in the elution chamber

42. The fourth pump 50 is also a bellows pump, and the nozzle portion of the fourth pump 50 is located in the elution chamber 42.

[0095] A heating element 58 is provided at the bottom surface of the elution chamber 42 for heating the contents of the elution chamber 42. The heating element 58 may be a variable heating element.

[0096] The elution chamber 42 includes an opening 100 at its top surface for accessing the contents of the elution chamber 42.

CA 02912883 2015-11-19 [0097] The second lid 54 is configured to provide access to the opening 100 in the top surface of the elution chamber 42. The second lid 54 is coaxial with the opening 100. The second lid 54 is shown being movably attached to the housing 12, such that when the lid 54 is open or off, the opening 100 is accessible and if the lid 54 is closed or on, the opening 100 is not accessible. [0098] With reference to Figure 2, as described above, the cassette 10 includes a housing 12. The housing 12 includes a first assembly component 102, a second assembly component 104 and a third assembly component 106.

[0099] The first assembly component 102 includes the mixing chamber 14, the washing chambers 38 and 40, the elution chamber 42 and the first stationary piece 86 of each of the valves 32,34 and 36. The first assembly component 102 also includes attachment parts 108,110 (see Figure 1) at one of its ends and an attachment piece 112 (see Figure 1).

[0100] The second assembly component 104 includes the holding chambers 16,18,20 and 22 and an opening for receiving the first pump 44. The second assembly component 104 also includes attachment receiving parts 114,116 (see Figure 1).

[0101] The third assembly component 106 indudes openings for receiving the second, third and fourth pumps 46,48 and 50, respectively, and indudes lids 52 and 54.

[0102] The cassette 10 is assembled by inserting the attachment components 108,110 of the first assembly component 102 into the attachment receiving components 114,116 of the second assembly component 104, respectively. The third assembly component 106 is then secured to the first assembly component using the attachment piece 112, thereby forming the assembled cassette 10, as illustrated in Figure 2. The plungers 24,26,28 and 30, pumps 44,46,48 and 50, and the second

CA 02912883 2015-11-19 moveable piece 88 of each of the valves 32,34 and 36, are inserted into the cassette 10.

[01031 In use, as shown in Figure 3, the first lid 52 is removed to provide access to the opening 70 of the mixing chamber 14. A sample of cells is placed into an assembled cassette 10 using a pipette 118. The cells in the sample include nucleic acid. The pipette 118 having the sample therein is placed in the mixing chamber 14. The sample is released from the pipette 118.

[0104] As shown in Figure 4, the cassette 10 is closed by closing the first lid

52. The cassette 10 is then placed together with similar cassettes 10 into a magazine 120, or rack, for containing a series of cassettes 10.

[01051 As shown in Figure 5, the magazine 120 is placed into an instrument 122. A protocol may be selected for preparing the sample in the cassette 10 in the instrument 122.

[0106] As shown in Figure 6, the PK solution is added to the sample. The PK solution is added by moving the plunger 30 in the fourth holding chamber 22. A force is applied to the base 78 of the plunger 30 to move the plunger 30. As the piercing element 82 of the plunger 30 advances toward the mixing chamber 14, the piercing element 82 punctures and ruptures the thin film 74. The break in the thin film 74 provides access to the mixing chamber 14. Continued motion of the plunger 30 transfers the contents (e.g., PK solution) of the first holding chamber 22 into the mixing chamber 14. [0107] The PK solution is mixed with the sample by pumping the mixture with the first pump 44. The PK solution destroys the walls of the cells of the sample, creating bulk material and nucleic acid in the bulk material.

[0108] As shown in Figure 7, the lysis solution is added to the sample. Plunger 28 operates in the same manner as plunger 30 to transfer the lysis solution in the third holding chamber 20 into the mixing chamber 14. The sample is pumped to mix the lysis buffer with the PK solution and sample of

CA 02912883 2015-11-19 cells. The lysis solution is typically a salt or detergent. The lysis solution is used to solulibize the bulk material. The lysis solution typically does not solulibize proteins.

[0109] The heating element 56 may be used to heat the lysis solution and sample. The heating element 56 may be controlled by the instrument 122. As described hereinabove, the temperature of the heating element 56 may be variable, and is selected to optimize the effectiveness of the lysis solution. [0110] As shown in Figure 8, the binding solution is added to the sample, PK solution and lysis buffer solution. Plunger 26 operates in the same manner as plunger 30 to transfer the binding solution in the second holding chamber 18 into the mixing chamber 14. The solution is pumped to mix the binding solution with the PK solution, lysis solution and sample. The binding solution is typically hydrophobic and increases salt in the solution. The binding solution causes the nucleic acid to be magnetically charged. [0111] As shown in Figure 9, the magnetic beads are added to the solution and pumped for about two minutes. Plunger 24 operates in the same manner as plunger 30 to transfer the lysis solution in the first holding chamber 18 into the mixing chamber 14. The magnetic beads bind to the magnetically charged nucleic acid.

[0112] As shown in Figure 10, the magnetic beads, together with the nucleic acid, are bound to the first valve 32. The removable positionable magnet 60 is placed in the first valve 32 and slid to a position in the first valve 32 to attract the magnetic beads, which are bound to the nucleic acid, from the mixing chamber 14 to the first valve 32.

[0113] As shown in Figure 11, the magnetic beads, together with the nucleic acid, are then moved from the mixing chamber 14 and received in the first washing chamber 38.

[0114] Figure 12 is a detailed view of the valves 32,34 36 36, illustrating the movement of the magnetic beads from the mixing chamber 14 to the first

CA 02912883 2015-11-19 washing chamber 38. As discussed above each of the valves 32,34 and 36 include a first stationary piece 86 and a second moveable piece 88, the second piece 88 being moveable relative to the first piece 86.

[0115] The magnet 60 is inserted into the opening 94 of the second piece

88. The magnet 60 is inserted to a position corresponding to the openings 90 and 92 of the first piece 86. The magnet 60 attracts the magnetic beads from the mixing chamber 14 through the opening 90 in the first piece 86 and into the cavity 98 in the second piece 88. The second piece 88 is rotated such that the magnetic beads are sealed in the cavity 98 of the second piece 88, between surfaces of the second piece 88 and the first piece 86. The second piece 88 is rotated past the surface 94 of the first piece 86, such that the cavity 98 is accessible in the opening 92 of the first piece 86. The magnet 60 is then removed from the opening 94 in the second piece 88 to release the magnetic beads from the cavity 98 in the second piece 88.

[0116] As shown in Figure 13, the magnetic beads and nucleic acid are then washed with the washing solution by pumping the solution with the second pump 46. The magnetic beads, together with the nucleic add, are then bound to the second valve 34 by inserting the magnet 60 into the second valve 34, as described above with reference to Figure 12.

10117] As shown in Figure 14, the magnetic beads, together with the nucleic acid, are then moved from the first washing chamber 38 to the second washing chamber 40 using the second valve 34. The second valve 34 transfers the magnetic beads and nucleic acid from the first washing chamber 38 to the second washing chamber 40, as described above with reference to Figure 12.

[0118] As shown in Figure 15, the magnetic beads and nucleic add are then washed with the washing solution a second time by pumping the solution with the third pump 48. The magnetic beads, together with the nucleic acid,

CA 02912883 2015-11-19

<img file="CA2912883C_D0002.tif" />

are then bound to the third valve 36 by positioning the magnet 60 in the third valve 36, as described above with reference to Figure 12.

[0119] As shown in Figure 16, the magnetic beads and nucleic add are then moved from the second washing chamber 40 to the elution chamber 42. Hie magnetic beads and nucleic acid are transferred from the second washing chamber 40 to the elution chamber 42 using the procedure described above with reference to Figure 12.

[0120] An elution buffer solution is then mixed with the magnetic beads and nucleic acid by pumping the solution with the fourth pump 50. The heating element 58 may be used to heat the elution buffer, magnetic beads and nucleic add. The heating element 58 may be controlled by the instrument 122. The temperature may be variable and may be selected to [0121] The magnetic beads alone are then bound again to the third valve 36 by positioning the magnet 60 in the third valve 36 as described above with reference to Figure 12.

[0122] As shown in Figure 17, the magnetic beads alone are then moved from the elution chamber 42 back into the second washing chamber 40, leaving the nucleic add in the elution chamber 42. The magnetic beads are transferred from the elution chamber 42 to the second washing chamber 40 using the procedure described above with reference to Figure 12.

[0123] As shown in Figure 18, the prepared sample of nucleic acid may be accessed using a second pipette 124. The second lid 54 is removed to provide access to the opening 100 in the elution chamber 42. The pipette 124 is inserted into the opening 100 and the prepared sample of nudeic add is withdrawn.

[0124] As shown in Figure 19, a multi-channel pipette 126 may be used to access a plurality of samples from a plurality of cassettes 10.

CA 02912883 2015-11-19 [0125] Figure 20 illustrates an alternative embodiment of the cassette 10. The cassette 10a illustrated in Figure 20 differs from the cassette 10 illustrated in Figure 1 in that the assembly component 104a includes a seal 130, the plungers 24a, 26a, 28a and 30a each include seals 132,134,136 and 138, respectively, and the valves 32a, 34a and 36a have a different arrangement, as discussed hereinafter.

[0126] Figure 21 illustrates the assembly component 104a in more detail. The assembly component 104a includes a seal 130. The illustrated seal 130 is a double elastomer, which extends along the circumference of the assembly component 104a.

[0127] Figure 22 illustrates the plunger 24a in more detail. The plunger 24a includes a seal 132. The illustrated seal 132 is also a double elastomer, which extends along the circumference of the plunger 24a. It will be appreciated that each of plungers 26a, 28a and 30a may also have a similar arrangement.

[0128] Figures 23 and 24 illustrate the valve 32a in more detail. It will be appreciated that valves 34a and 36a also have a similar arrangement. The valve 32a includes a magnet 60a, a housing 142, and a shaft 144. The housing 142 includes a first opening (not shown) to receive the magnet 60a and a second opening 148 to expose the magnet 60a and receive the particles 146. The magnet 60a is shaped to correspond to the opening 148 and is selected to attract the particles 146. The housing 142 also includes a third opening (not shown) for receiving the shaft 144. As shown in Figure 24, the shaft 144 may include a keyed element 150. Tlie keyed element 150 is shaped to engage the cassette 10a. It will be appreciated that the shaft may be removable or an integrated element of the valve 32a. It will also be appreciated that the housing 142 may, alternatively, include the keyed element.

CA 02912883 2015-11-19 [0129] The shaft 144 is engageable with the housing 142 and magnet 60a to rotate the housing 142 and magnet 60a relative to the cassette 10a to move the particles 146 from the mixing chamber 14a to the washing chamber 38a. It will be appreciated that valves 34a and 36a operate in a similar manner to transfer the particles 146 from the washing chamber 38a to the washing chamber 40a and from the washing chamber 40a to the elution chamber 42a, respectively.

[0130] In one embodiment, a total of about 200 gL sample is placed into the cassette. The sample is mixed with a total of about 50 gL of the PK solution by pumping the mixture of the sample and PK solution for about one minute. A total of about 200 gL of the lysis solution is added to the sample and PK solution, and the solutions are pumped for about one minute to mix the solutions. The mixture is then heated at about 60°C for about ten minutes, and the mixture is allowed to cool for about 5 minutes. The mixture is further pumped while it cools. A total of about 500 gL of binding solution is added to the mixture. The solutions are pumped for about one minute. The magnetic beads are added to the solution and pumped for about two minutes. The magnetic beads are transferred and washed as described above. A total of about 700 gL of washing solution is provided in each of the washing chambers. A total of about 200 gL of elution solution is provided in the elution chamber. The magnetic beads are mixed with the elution solution by pumping the mixture for about one minute. The mixture is then heated at about 90°C for about two minutes. The process continues as previously described.

[0131] Although the cassette 10 has been described as having a mixing chamber 14, two washing chambers 38 and 40 and an elution chamber 42, it is envisioned that only one washing chamber or no washing chamber may alternatively be provided.

CA 02912883 2015-11-19 [0132] Although the cassette has been described as using a single removable magnet 60, it is envisioned that each valve may include a positionable magnet, such that the magnet does not need to be removed. The magnet 60 may be rotatable, and used to rotate the second piece of the valves. Alternatively, the magnet may only slide inside of each of the valves, and the second piece is rotated independent of the magnet.

[0133] It is envisioned that a cassette 10 that does not use valves as described herein may be used to transfer the magnetic particles from the mixing chamber to the elution chamber. In such an embodiment, a slideable magnet may be provided to transfer the magnetic particles from one chamber to the next.

[0134] Although the cassette 10 has been described as using a PK solution, lysis solution, binding solution and magnetic beads to release the nucleic add and magnetic beads, it is envisioned that it may be possible to practice the invention without using each of the above solutions. In addition, although the solution was described as using a PK solution to break up the cells, it is envisioned that any enzyme which causes cells to break up to release nucleic add may be used with the invention.

[0135] It is envisioned that the housing 12 may be transparent, such that the procedure can be viewed.

[0136] In one embodiment the thin film 74 is a lamination.

[0137] In one embodiment, the lids 52 and 54 may be screw-top lids. In one embodiment, the lids 52,54 include a hydrophobic membrane, which allows gasses to vent through the lid, but does not allow the liquids to escape the cassette 100.

[0138] In one embodiment, pump 50 is insertable into opening 100. In one embodiment, pump 50 can also be used as a pipette to remove the sample from the cassette 10.

CA 02912883 2015-11-19 [0139] It is also envisioned that the mixing chamber 14 may be provided without a puncturable thin film 74. In such an embodiment, the plungers 24,26,28 and 30 would not need a piercing element 82. Instead, the plungers 24,26,28 and 30 would have a sealing element to prevent leakage of the contents of the holding chamber 16,18,20 and 22, associated with each plunger 24,26,28 and 30, respectively, until the plunger was moved. [0140] Figure 25 illustrates a cassette 200, which can be used to prepare cell samples. The cassette 200 includes a housing 202, first, second, third, fourth, fifth, sixth, seventh and eighth holding chambers 204a-h, respectively. Each of the holding chambers 204a-h includes a valve assembly 206a-h therein. A locking element 207 may also be provided.

[0141] Figure 26 illustrates the cassette 200 in more detail. The cassette 200 further includes a reaction chamber 208, a particle chamber 210, a waste chamber 212, a waste overflow chamber 214, an elution chamber 216, a plunger 218, and first and second lids 220 and 222, respectively. The cassette 200 may also include one or more heating elements (not shown). [0142] Each of the holding chambers 204a-h, valve assemblies 206a-h, reaction chamber 208, particle chamber 210, waster chamber 212, waster overflow chamber 214, and plunger 218 are enclosed within the housing 202. The lids 222,224 are movably or removably attached to the housing 202. [0143] The reaction chamber 208 has a top surface 226, a bottom surface 228 and opposing side surfaces 230,232.

[0144] The top surface 226 of the reaction chamber 208 includes an opening 234 therein. The first lid 222 is configured to provide access to the opening 234 in the top surface 226 of the reaction chamber 208. The illustrated lid 222 is a screw-top lid; however, any other lid which (removably) provides access to the opening 234.

CA 02912883 2015-11-19 [0145] The bottom surface 228 of the reaction chamber 208 includes an opening 236 therein. The opening 236 allows the reaction chamber 208 to be in fluid communication with the particle chamber 210.

[0146] The side surface 232 includes openings 238a-h therein. The openings 238a-h allow the reaction chamber 208 to be in fluid communication with the holding chambers 204a-h, respectively.

[0147] The cassette 200 includes a binding solution in a holding chamber 204a. The cassette 200 further includes a lysis solution in a holding chamber 204b. The cassette 200 further includes a proteinase K (PK) solution in a holding chamber 204c. The cassette 200 further includes a washing solution in one or more of the holding chambers 204d-e. The cassette 200 further includes an elution solution in a holding chamber 204f.

[0148] The plunger 218 and the first lid 222 are shown attached to one another to form an integral plunging system. The plunger 218 is compressible to pump the contents of the reaction chamber 208. Alternatively, a separate pump may also be provided to pump the contents of the reaction chamber 208. The plunger 218 is also moveable within the reaction chamber 208 to push the contents of the reaction chamber 208 through the particle chamber 210.

[0149] The holding chambers 204a-h are formed in the housing 202 of the cassette 200. Each of the holding chambers 204a-h includes a guide 240a-h engageable with a corresponding slot in the valve assembly 206. The holding chambers 204a-h also include at least one opening 242a-h, engageable with corresponding openings in the valve assembly 206a-h. The housing 202 also includes slots 244a-h, engageable with corresponding guides in the valve assembly 206a-h.

[0150] The particle chamber 210 includes a body 250, having a first opening 252, a second opening 254, and a plurality of particles 256 thereon. The particles may be magnetic or nonmagnetic, depending on the

CA 02912883 2015-11-19 application of the cassette 200. The particles may be, for example, cellulose, plastic or iron. The particle chamber 210 is shown aligned with the reaction chamber 208.

[0151] The waste chamber 212 and the elution chamber 216 are integrated with one another and are rotatable relative to the housing 202. The waste overflow chamber 214 is positioned near the waste chamber 212 and is capable of being in fluid communication with the waste chamber 212. The waste chamber 212 and elution chamber 216 are alignable with the particle chamber and are capable of being in fluid communication with the particle chamber 210.

[0152] The waste chamber 212 has a top surface 270, a bottom surface 272, an inner surface 274 and an outer surface 276. The overflow waste chamber 214 has a top surface 278, a bottom surface 280 and opposing side surfaces 282,284. The elution chamber 216 also has a top surface 286, a bottom surface 288, an inner surface 290 and an outer surface 292.

[0153] It will be appreciated that the outer surface 276 of the waste chamber 212 and the outer surface 292 of the elution chamber 216 are integrated with one another. It will also be appreciated that the inner surface 274 of the waste chamber 212 is the same as the inner surface 290 of the elution chamber 216.

[0154] The top surface 270 of the waste chamber 212 and the top surface 286 of the elution chamber 216 each have an opening 294,296, respectively. These openings 294,296 are alignable with the opening 252 in the particle chamber 210 to provide a fluid communication route between the particle chamber 210 and the waste chamber 212 and the elution chamber 216.

[0155] The outer surface 276 of the waste chamber 212 includes an opening 298 therein. One of the side surfaces 282,284 of the overflow waste chamber 214 includes an opening 300 therein. The opening 298 and opening 300 are

CA 02912883 2015-11-19 alignable, such that fluid flowing into the waste chamber 212 can flow from the waste chamber 212 and into the overflow waste chamber 214.

[0156] The bottom surface 288 of the elution chamber 216 includes an opening 302 therein. The second lid 224 is configured to provide access to the opening 302 in the bottom surface 288 of the elution chamber 216. The illustrated lid 224 is a screw-top lid; however, any other lid which (removably) provides access to the opening 302.

[0157] Figures 27 and 28 illustrate the valve assembly 206 in more detail. Valve assembly 206 includes a housing 310. The housing 310 includes a chamber 312 therein, a slot 314, and projections 316 extending therefrom. The chamber 312 includes a pump 318 therein. A lid 320 is provided at an end of the housing to seal the chamber 310. The lid includes first and second openings 322,324, extending therethrough and providing fluid communication with the chamber 312.

[0158] When the openings 322,324 are not aligned with openings 238a-h, the contents of the chamber 312 are sealed within the valve assembly; however, when the openings 322,324 are aligned with openings 238a-h, the contents of the chamber 312 are releasable from the chamber 312. The slot 314 is used to guide the openings 322,324 to the location where the openings 322,324 are aligned with the openings 238a-h. The pump 318 is used to transfer the contents from the chamber 312 and into the reaction chamber 214 through the openings 322,324.

[0159] The cassette 200 is assembled by inserting the valve assemblies 206a-h into the holding chambers 204a-h of the housing. The locking element 207 may be connected to the housing to secure valve assemblies 206a-h in the holding chambers 204a-h. The waste chamber and elution chamber 216 assembly is inserted into the housing 202 and the lid 224 is secured to the housing 202. The plunger 218 is also inserted into the reaction chamber 208 and the lid 222 is secured to the housing.

CA 02912883 2015-11-19 [0160] In use, as shown in Figure 29, the first lid 222 is removed to provide access to the opening 234 of the reaction chamber 208. A sample of cells 350 is placed into the cassette 200 using a pipette 352. The cells in the sample include nucleic acid. The pipette 352 having the sample therein is placed in the reaction chamber 208. The sample is released from the pipette 352.

[0161] As shown in Figure 30, the PK solution is added to the sample. The PK solution is added by rotating the valve assembly 206c relative to the housing 202. At least one of the openings 322,324 of the valve assembly 206c is aligned with the opening 242c in the holding chamber 204c to release the PK solution from the holding chamber 204c and into the reaction chamber 208 through the opening 238c in the housing 202.

[0162] The PK Solution is mixed with the sample by pumping the mixture with the plunger 218. As described hereinabove, the PK solution destroys the wall so the cells of the sample, creating bulk material and nucleic acid in the bulk material.

[0163] As shown in Figure 31, the lysis solution is added to the sample. The valve assembly 206b operates in the same manner as valve assembly 206c to transfer the lysis solution in the holding chamber 204b into the reaction chamber 208. The sample is typically pumped to mix the lysis solution with the mixed PK solution and sample. The lysis solution is typically a salt or detergent, and is used to solulibize the bulk material, as discussed hereinabove.

[0164] As shown in Figure 32, a binding solution is added to the sample, PK solution and lysis solution. Valve assembly 206a operates in the same manner as valve assembly 206c to transfer the binding solution in the holding chamber 204a into the reaction chamber 208. The solution is pumped to mix the binding solution with the PK solution, lysis solution and sample. The binding solution is typically hydrophobic and increases sale in the solution.

CA 02912883 2015-11-19 [0165] As shown in Figure 33, the solution is pumped through the particles in the particle chamber 210. The nucleic add binds to the particles in the particle chamber, while the remaining solution flows into the waste chamber 212, and, if needed, the waste overflow chamber 214. As described above, when the opening 298 in the waste chamber 212 is aligned with the opening 300 in the waste overflow chamber 214, the solution can flow into the waste overflow chamber 214 from the waste chamber 212.

[0166] As shown in Figure 34, the washing solution is added to the reaction chamber 208 by operating the valve assembly 206d in the same manner as valve assembly 206c to transfer the washing solution from the holding chamber 204d into the reaction chamber 208.

[0167] As shown in Figure 35, the first washing solution is pumped through the particle chamber 210 and into the waste chamber 212, and, if needed, the waste overflow chamber 214.

[0168] As shown in Figure 36, a second washing solution is added to the reaction chamber 208 by operating the valve assembly 206e in the same manner as valve assembly 206c to transfer the washing solution from the holding chamber 204e into the reaction chamber 208.

[0169] As shown in Figure 37, the second washing solution is pumped through the partide chamber 210 and into the waste chamber 212, and, if needed, the waste overflow chamber 214.

[0170] As shown in Figure 38, an elution solution is added to the reaction chamber 208 by operating the valve assembly 206f in the same manner as valve assembly 206c to transfer the elution solution from the holding chamber 204f into the reaction chamber 208.

[0171] The waste chamber and elution chamber assembly is rotated to

<img file="CA2912883C_D0003.tif" />

particle chamber 210 such that the elution solution is transferable into the elution chamber 216. It will be appredated that the opening 296 can be

CA 02912883 2015-11-19 aligned with the opening 252 before or after the elution solution is added to the reaction chamber 208.

[0172] As shown in Figure 39, the elution solution is pumped through the particle chamber 210 to elute the bound nucleic add. The nucleic add and elution solution flow into the elution chamber 216.

[0173] As shown in Figure 40, the prepared sample of nudeic add may be accessed using a pipette 354. The second lid 224 is removed to provide access to the opening 302 in the elution chamber 216. The pipette 354 is inserted into the opening 302 and the prepared sample of nucleic acid is withdrawn.

[0174] It will be appreciated that the cassette 200 can be placed with similar cassettes into a magazine or rack for containing a series of cassettes. The magazine or rack can be placed into an instrument, and a protocol may be selected for preparing the sample in the cassette 200 in the instrument.

[0175] Cassette 200 may include one or more heating elements as described hereinabove with respect to cassette 100.

[0176] The cassettes 100,200 may be disposable.

[0177] It will be appreciated that although the cassettes 100,200 have been described with respect to breaking cells to extract nucleic add, the cassettes 100,200 can be used to break cells to extract other cell components, such as, for example, protein. Also, although a lysis solution has been described as being used to break up cells, it will be appredated that any substance that can break up cells, such as, for example, reagents, enzymes, catatropic salts, other lysis solutions and the like.

[0178] The cassettes described herein are advantageous because it is dosed. There is no contamination of the sample during the process. In addition, a fewer number of samples, including as few as one sample, may be prepared.

CA 02912883 2015-11-19

The foregoing description with attached drawings is only illustrative of possible embodiments of the described method and should only be construed as such. Other persons of ordinary skill in the art will realize that many other specific embodiments. The abovedescribed embodiments are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art without departing from the scope, which is defined solely by the claims appended hereto.