EP2241613A1

EP2241613A1 - Fluid receiving cartridge and utilization of the same

Info

Publication number: EP2241613A1
Application number: EP08862757A
Authority: EP
Inventors: Masahiro Murasato; Kazunari Yamada; Dai Yamamoto; Akinobu Oribe; Kousuke Niwa
Original assignee: NGK Insulators Ltd
Current assignee: NGK Insulators Ltd
Priority date: 2007-12-14
Filing date: 2008-12-12
Publication date: 2010-10-20
Also published as: JP2009240296A; CN101896598A; US20100276309A1; WO2009078352A1; EP2241613A4

Abstract

In a state where liquids are contained in a plurality of liquid container portions 1-15 and 17 which are formed in a first layer 50a and a second layer 50b of a cartridge and which have predetermined volumes determined depending on liquids to be contained, and where the reaction vessel is connected to any one of communicating ports 1a-16a, when pressure is applied to act upon one of the liquid container portions which is communicated with the communicating port connected to the reaction vessel, the atmosphere is supplied to the one liquid container portion through a atmosphere flowing passage 21 communicating with the outside, and the liquid contained in the one liquid container portion is supplied to the reaction vessel. The cartridge is rotated to connect another communicating port to the reaction vessel such that plural liquids are eventually supplied to the reaction vessel. On that occasion, since the atmosphere supplied through the atmosphere flowing passage 21 is further supplied to the reaction vessel through the communicating port connected to the reaction vessel and the one liquid container portion, the plural liquids contained in the reaction vessel are stirred by the inflow of the atmosphere.

Description

Technical Field

The present invention relates to a fluid containing cartridge, a fluid reaction unit, a gene analyzing method using the fluid containing cartridge, a reaction apparatus using the fluid containing cartridge, and a reaction method using the reaction apparatus.

Background Art

In a hitherto known fluid containing cartridge, the cartridge is constituted as a container formed by a base plate made of a rigid member and by an elastic member, and a plurality of chambers are formed in the container such that the chambers are coupled with each other or arranged to be capable of being coupled with each other through flow passages (see, e.g., Patent Document 1). In the fluid containing cartridge described in Patent Document 1, fluid materials in the flow passages and the chambers are moved so as to develop chemical reactions upon application of an external force to the elastic member from the outside of the container.

Patent Document 1: JP 2005-313065 A

Disclosure of Invention

However, the fluid containing cartridge described in Patent Document 1 has a problem that a plurality of fluids to be subjected to chemical reactions cannot be fully mixed with each other and the chemical reactions cannot be sufficiently developed in some cases, for example, when the external force is applied by using such a simple mechanism as moving the fluids in one direction while pressing a roller.
The present invention has been made in view of the above-described problem, and a main object of the present invention is to provide a fluid containing cartridge, a fluid reaction unit, a gene analyzing method using the fluid containing cartridge, a reaction apparatus using the fluid containing cartridge, and a reaction method using the reaction apparatus, the fluid containing cartridge enabling contained fluids to sufficiently develop a reaction in a reaction vessel.
The present invention employs the following means to achieve the above object.
According to the present invention, there is provided a fluid containing cartridge for containing fluids in an extractable manner, the fluid containing cartridge comprising:

a plurality of container portions being able to contain the fluids and formed inside a housing in respective predetermined volumes that are determined depending on the fluids to be contained therein;
an atmosphere flowing portion for communicating the container portions with the outside, thus enabling the atmosphere to flow into the container portions; and
a plurality of connecting and communicating portions disposed at respective predetermined connecting positions where, by relatively moving a reaction vessel disposed in the outside and capable of containing the fluids and the fluid containing cartridge in a selective manner, the reaction vessel and any one of the container portions are connected to each other for fluid communication therebetween, the connecting and communicating portions enabling the fluids contained in the container portions to be supplied to the reaction vessel by differential pressure acting upon the fluids contained in the container portions.

According to the fluid containing cartridge mentioned above, in a state where the fluids are contained in the plurality of container portions which are formed in the housing and which have predetermined volumes determined depending on the liquids to be contained, and where the reaction vessel is connected to any one of the connecting and communicating portions, when differential pressure is applied to act upon one of the container portions, the atmosphere is supplied to the one liquid container portion through the atmosphere flowing passage communicating with the outside, and the liquid contained in the one liquid container portion is supplied to the reaction vessel. Further, the fluid containing cartridge is moved to connect another connecting and communicating portion to the reaction vessel such that plural liquids are eventually supplied to the reaction vessel. On that occasion, since the atmosphere supplied through the atmosphere flowing passage is further supplied to the reaction vessel through the one container portion and the connecting and communicating portion, the plural liquids contained in the reaction vessel are subjected to bubbling with the inflow of the atmosphere. Accordingly, the contained liquids can be caused to sufficiently develop a reaction in the reaction vessel.
In the fluid containing cartridge according to the present invention, the plurality of connecting and communicating portions may be disposed on a flat surface formed in the housing in a circular pattern coaxial with an axis of rotation about which any one of the reaction vessel and the housing is rotated. With that feature, when the plurality of connecting and communicating portions are selectively connected to the reaction vessel, it is just required to rotationally move the reaction vessel or the cartridge. Therefore, the connection can be selectively changed with ease and the fluids can be easily introduced to the reaction vessel, thus enabling a reaction to be sufficiently developed with ease. In that case, at least one of the plurality of container portions may be formed in the housing having the shape of a circular disk and may have the shape of a zigzag tube with a zigzag width gradually increasing from an inner peripheral side toward an outer peripheral side of the circular disk-shaped housing. Further, the plurality of connecting and communicating portions may be disposed in the inner peripheral side of the housing having the shape of a circular disk. A space in the housing having the shape of a circular disk can be effectively utilized by gradually increasing the zigzag width toward the outer peripheral side, and the fluid contained in the container portion can be fully supplied to the reaction vessel by forming the container portion into the tube-like shape. As an alternative, the plurality of container portions may be formed in the housing having the shape of a circular disk such that the container portions containing the fluids in larger amounts are positioned in an even outer peripheral side of the circular disk-shaped housing. That feature makes it easier to form the container portions containing the fluids in larger amounts.
In the fluid containing cartridge according to the present invention, at least one of the plurality of container portions may be formed in the shape of a tube gradually narrowing toward the connecting and communicating portion. That feature is effective in more easily transferring all the fluid from the container portion to the reaction vessel and in more satisfactorily developing the reaction.
In the fluid containing cartridge according to the present invention, at least one of the plurality of container portions may be formed in the shape of a tube gradually narrowing toward opposite ends thereof. That feature is effective in preventing the contained fluid from flowing out when the fluid containing cartridge is distributed or handled, and in causing the contained fluid to more satisfactorily develop the reaction in the reaction vessel.
In the fluid containing cartridge according to the present invention, the plurality of container portions may be formed such that the container portions containing the fluids in larger amounts have the shapes of longer tubes. That feature enables the container portions to more efficiently contain the fluids.
In the fluid containing cartridge according to the present invention, the housing may comprise a plurality of divided layers, and the container portions may be formed in any one of the divided layers, or formed in a state extending over two or more of the divided layers. With that feature, a larger number of container portions can be formed by using the divided layers. In that case, the plurality of container portions may be formed such that the container portions formed in the divided layer positioned farther away from the flat surface, in which the connecting and communicating portions are formed, are able to contain the fluids in larger amounts. With that feature, since the number of container portions reduces in the side farther away from the connecting and communicating portions, the number of paths for connecting the container portions to the connecting and communicating portions is reduced and the cartridge is easier to fabricate.
The fluid containing cartridge according to the present invention may further comprise a reservoir portion being able to reserve a fluid and formed in the housing to be communicated with the atmosphere, and a coupling and communicating portion disposed at a predetermined coupling position where the reaction vessel and the reservoir portion are communicated with each other when the reaction vessel and the fluid containing cartridge are relatively moved into a state of predetermined positional relation in a selective manner. With that feature, a reaction requiring the fluid(s) in the reaction vessel to be reserved can also be developed. In such a case, the fluid containing cartridge may further comprise an outflow restriction portion disposed between the reservoir portion and the coupling and communicating portion to allow flow of the fluid from the reaction vessel to the reservoir portion and to block off flow of the fluid from the reservoir portion to the reaction vessel, a column portion disposed between the coupling and communicating portion and the outflow restriction portion and being able to adsorb a product produced in the reaction vessel, and an inflow restriction portion disposed between any one of the plurality of container portions and the column portion to allow flow of the fluid from the one container portion to the column portion and to block off flow of the fluid from the column portion to the one container portion. With that feature, after adsorbing in the column the product that has been produced with the reaction developed in the reaction vessel, it is possible to supply the fluid in the container portion in a way flowing through the column portion and to return the relevant fluid to the reaction vessel without changing the connection between the reaction vessel and the coupling and communicating portion. In such a case, the column portion may include a ceramic column. Additionally, the above-described construction of the container portion may also be applied to the reservoir portion.
In the fluid containing cartridge including the reservoir portion, according to the present invention, an absorbing material for absorbing the fluid may be disposed in the reservoir portion. That feature is effective in more positively holding the fluid in the reservoir portion once the fluid has been introduced to the reservoir portion.
In the fluid containing cartridge according to the present invention, the container portions may contain, as the fluids, liquids necessary for amplifying, fragmentizing, ligation-causing or labeling SNP and genome DNA for a variation analysis, and liquids necessary for reverse-transcribing cDNA from RNA and amplifying, fragmentizing, ligation-causing or labeling the cDNA. The present invention has high significance when applied to the field of biotechnology because of the necessity of not only preparing plural kinds of liquids for amplifying, fragmentizing, ligation-causing or labeling SNP and genome DNA for a variation analysis, and plural kinds liquids for reverse-transcribing cDNA from RNA and amplifying, fragmentizing, ligation-causing or labeling the cDNA, but also causing the liquids to sufficiently develop the reaction.
In the fluid containing cartridge according to the present invention, the container portions may contain, as the fluids, liquids necessary for amplifying, fragmentizing or labeling genome DNA for a chromosome anomaly analysis. The present invention has high significance when applied to the field of biotechnology because of the necessity of not only preparing plural kinds of liquids for amplifying, fragmentizing or labeling genome DNA for a chromosome anomaly analysis, but also causing the liquids to sufficiently develop the reaction.
In the fluid containing cartridge according to the present invention, the atmosphere flowing portion may comprise an atmosphere flowing passage having an atmosphere hole and communicating at least one of the container portions with the outside, and a porous material disposed in the atmosphere flowing passage and allowing passing of the atmosphere, but not allowing passing of any fluid. With that feature, the fluid contained in the container portion can be prevented from flowing out to the outside through the atmosphere flowing portion. In such a case, the atmosphere flowing portion may be connected to two or more of the container portions. With that feature, it is possible not only to prevent the fluid contained in the container portion from flowing out to the outside through the atmosphere flowing portion, but also to reduce the number of atmosphere holes communicating with the outside.
In addition, the connecting and communicating portion may be connected to the reaction vessel such that the fluid(s) contained in the reaction vessel can be supplied to the container portion by differential pressure acting upon the fluid(s) contained in the reaction vessel. With that feature, a new reaction, etc. can be performed in the reaction vessel by causing the fluid contained in the reaction vessel to be introduced to the container portion.
A fluid reaction unit according to the present invention comprises:

any one of the fluid containing cartridges described above; and
a reaction vessel being connectable to the plurality of connecting and communicating portions disposed in the fluid containing cartridge and being able to contain the fluid supplied through the connecting and communicating portion connected.

Since the fluid reaction unit includes any one of the fluid containing cartridges described above, it can provide the advantages obtained with any one of the fluid containing cartridges described above, such as the advantage of enabling the contained fluids to sufficiently develop the reaction in the reaction vessel, and the advantage that when the plurality of connecting and communicating portions are selectively connected to the reaction vessel, it is just required to rotationally move the reaction vessel or the cartridge, whereby the connection can be selectively changed with ease and the fluids can be easily introduced to the reaction vessel, thus causing a reaction to be sufficiently developed with ease. In such a case, the reaction vessel may be formed in the shape of a tube gradually narrowing toward the connecting and communicating portion. That feature is effective in causing all the fluid to easily flow between the fluid container portion and the reaction vessel through the connecting and communicating portion, and in easily bubbling the fluid(s) in the reaction vessel by the atmosphere introduced through the connecting and communicating portion.
According to the present invention, there is provided a reaction apparatus for mixing a plurality of fluids with each other to develop a reaction, the reaction apparatus comprising:

mounting unit capable of mounting any of the fluid containing cartridges described above;
a reaction vessel being connectable to the plurality of connecting and communicating portions disposed in the mounted fluid containing cartridge and being able to contain the fluid supplied through the connecting and communicating portion connected;
a moving unit for moving at least one of the reaction vessel and the mounted fluid containing cartridge to a predetermined connecting position where the reaction vessel and any one of the plurality of connecting and communicating portions are connected to each other; and
a pressure applying unit capable of applying differential pressure to act upon the container portion of the fluid containing cartridge, thereby supplying the fluid contained in the container portion to the reaction vessel.

In the reaction apparatus, after mounting any one of the fluid containing cartridges described above, at least one of the reaction vessel and the mounted fluid containing cartridge is moved to the predetermined connection position where the reaction vessel is connected to any one of the plurality of connecting and communicating portions disposed in the mounted fluid containing cartridge, and differential pressure is applied to act upon one of the container portions of the fluid containing cartridge such that the fluid contained in the one container portion is supplied to the reaction vessel. Thus, when the differential pressure is applied to act upon the container portion in the state where the reaction vessel is connected to the connecting and communicating portion, the atmosphere is supplied to the container portion through the atmosphere flowing passage communicating with the outside, and the fluid contained in the container portion is supplied to the reaction vessel. Further, the fluid containing cartridge is moved to connect another connecting and communicating portion to the reaction vessel such that plural fluids are eventually supplied to the reaction vessel. On that occasion, since the atmosphere supplied through the atmosphere flowing portion is further supplied to the reaction vessel through the container portion and the connecting and communicating portion, the plural fluids contained in the reaction vessel are subjected to bubbling by the inflow of the atmosphere. As a result, the contained fluids can be caused to more satisfactorily develop the reaction in the reaction vessel.
The reaction apparatus according to the present invention may be a reaction apparatus for mixing a plurality of fluids with each other to develop a reaction, the reaction apparatus comprising:

a connecting unit capable of connecting any of the fluid reaction units described above;
a moving unit for moving at least one of the reaction vessel in the connected fluid reaction unit and the mounted fluid containing cartridge to a predetermined connecting position where the reaction vessel and any one of the plurality of connecting and communicating portions are connected to each other; and
a pressure applying unit capable of applying differential pressure to act upon the container portion of the fluid containing cartridge, thereby supplying the fluid contained in the container portion to the reaction vessel.
In that reaction apparatus, the contained fluids can also be caused to more satisfactorily develop the reaction in the reaction vessel.

In the reaction apparatus according to the present invention, the reaction vessel may be formed in the shape of a tube gradually narrowing toward the connecting and communicating portion. That feature is effective in causing all the fluid to easily flow between the fluid container portion and the reaction vessel through the connecting and communicating portion, and in easily bubbling the fluid(s) in the reaction vessel by the atmosphere introduced through the connecting and communicating portion.
The reaction apparatus according to the present invention may further comprise a control unit for, in accordance with a series of reaction procedures to be executed by using the fluids contained in the container portions of the mounted fluid containing cartridge, controlling the moving unit such that one of the plurality of container portions is selectively connected in turn to the reaction vessel, and controlling the pressure applying unit to transfer the fluid by the action of the differential pressure applied to the container portion.
In the reaction apparatus according to the present invention, the pressure applying unit may be connected to the reaction vessel to suck the fluid from the container portion into the reaction vessel by lowering air pressure in the reaction vessel and to push out the fluid from the reaction vessel to the container portion by raising air pressure in the reaction vessel. With that feature, since the fluid can be sucked from the container portion and pushed out to the container portion just by changing the pressure in the reaction vessel, the fluid can be transferred with a comparatively simple construction.
In the reaction apparatus according to the present invention, the mounting unit may mount the fluid containing cartridge provided with a circular disk-shaped housing in which the container portions are formed, and the moving unit may move at least one of the reaction vessel and the mounted fluid containing cartridge by rotationally moving one of the reaction vessel and the fluid containing cartridge about an axis of rotation. With that feature, since it is just required to rotationally move the reaction vessel or the cartridge when the plurality of connecting and communicating portions are selectively connected to the reaction vessel, the connection can be changed with more ease. In such a case, the pressure applying unit may act pressure upon the container portion of the fluid containing cartridge through the reaction vessel, and the moving unit may rotationally move the fluid containing cartridge about the axis of rotation. With that feature, the fluid containing cartridge can be easily rotated because the pressure applying unit is not connected to the fluid containing cartridge. The mounting unit may mount the fluid containing cartridge by using a contact member disposed on a flat surface of the housing having the shape of a circular disk, in which the connecting and communicating portions are formed, and further disposed in a circular shape coaxial with the axis of rotation about which the housing is rotationally moved, the contact member contacting with the flat surface of the fluid containing cartridge and having a through-hole communicating with the connecting and communicating portion, and by using a fixing member for rotatably fixing the fluid containing cartridge in a state of the contact member being held in contact with the flat surface of the fluid containing cartridge, and the reaction vessel may be connected to the through-hole formed in the contact member. With that feature, the fluid containing cartridge can be mounted in such a state that the container portion and the reaction vessel can be comparatively easily connected to each other in a selective manner.
The reaction apparatus according to the present invention may further comprise a cartridge temperature adjusting unit capable of adjusting temperature of the fluid containing cartridge mounted to the mounting unit, and a reaction-vessel temperature adjusting unit capable of adjusting temperature of the reaction vessel. That feature makes it possible to separately adjust the mounted fluid containing cartridge to temperature not causing the contained liquids to develop the reaction and the reaction vessel to temperature suitable for the reaction. As a result, the liquids contained in the fluid containing cartridge can be caused to sufficiently develop the reaction in the reaction vessel regardless of the temperature of the mounted fluid containing cartridge.
According to the present invention, there is provided a reaction method using a reaction apparatus for mixing a plurality of fluids with each other to develop a reaction, the reaction method comprising the steps of:

(a) preparing the fluid containing cartridge according to any one of Claims 1 to 16, which is mounted to the reaction apparatus, and a reaction vessel which is connectable to the plurality of connecting and communicating portions disposed in the mounted fluid containing cartridge and which is able to contain the fluid supplied through the connecting and communicating portion connected to the reaction vessel, and moving at least one of the reaction vessel and the mounted fluid containing cartridge to a predetermined connecting position where the reaction vessel and any one of the plurality of connecting and communicating portions are connected to each other; and
(b) applying pressure to the container portion of the fluid containing cartridge to introduce the fluid contained in the relevant container portion to the reaction vessel.

With the reaction method, at least one of the reaction vessel and the mounted fluid containing cartridge is moved to the predetermined connecting position where the reaction vessel and any one of the plurality of connecting and communicating portions are connected to each other, the reaction vessel being connectable to the plurality of connecting and communicating portions disposed in one of the fluid containing cartridges described above, which is mounted to the reaction apparatus, and being able to contain the fluid supplied through the connecting and communicating portion connected to the reaction vessel. Further, pressure is applied to the container portion of the fluid containing cartridge to introduce the fluid contained in the relevant container portion to the reaction vessel. Thus, when differential pressure is applied to act upon the container portion in the state where the reaction vessel is connected to the connecting and communicating portion, the atmosphere is supplied to the container portion through the atmosphere flowing passage communicating with the outside, and the fluid contained in the container portion is supplied to the reaction vessel. Further, the fluid containing cartridge is moved to connect another connecting and communicating portion to the reaction vessel such that plural fluids are eventually supplied to the reaction vessel. On that occasion, since the atmosphere supplied through the atmosphere flowing portion is further supplied to the reaction vessel through the container portion and the connecting and communicating portion, the plural fluids contained in the reaction vessel are subjected to bubbling by the inflow of the atmosphere. As a result, the contained fluids can be caused to more satisfactorily develop the reaction in the reaction vessel.
A gene analyzing method according to the present invention is to detect SNP, variation of DNA, or the representation (abundance) of RNA (e.g., mRNA or miRNA) by using the fluid containing cartridge which contains in its container portions, as the fluids, liquids necessary for amplifying, fragmentizing, ligation-causing or labeling SNP and genome DNA for a variation analysis, and liquids necessary for reverse-transcribing cDNA from RNA and amplifying, fragmentizing, ligation-causing or labeling the cDNA.
The gene analyzing method according to the present invention is to measure change in the number of chromosome copies for a human chromosome by using the fluid containing cartridge which contains, as the fluids, liquids necessary for amplifying, fragmentizing, or labeling genome DNA for a chromosome anomaly analysis.

Brief Description of Drawings

Fig. 1 is an overall view illustrating the general construction of a reaction apparatus 90.
Fig. 2 is an explanatory view to explain a cartridge mounting mechanism 80 and a cross-section after mounting a cartridge.
Fig. 3 is an explanatory view illustrating degassing grooves formed in a reaction vessel 30.
Fig. 4 is an explanatory view illustrating chambers and flow passages inside a cartridge 50.
Fig. 5 is an explanatory view illustrating a micro check valve 160.
Fig. 6 is an explanatory view illustrating the position of a porous material inside the cartridge 50.
Fig. 7 is an explanatory view illustrating a heat-insulated structure of the reaction apparatus 90.
Fig. 8 is a diagram to explain procedures for obtaining cDNA from mRNA in the reaction apparatus 90.
Fig. 9 is an explanatory view illustrating a flow route when a mixed solution is caused to flow toward a column portion 16.
Fig. 10 is an explanatory view illustrating a flow route when an elution buffer is sucked out through the column portion 16.
Fig. 11 is an explanatory view illustrating chambers and flow passages inside a cartridge 150.
Fig. 12 is a diagram to explain procedures for producing genome DNA labeled for detection of SNP.
Fig. 13 is an explanatory view illustrating connection relations among a column 106, the reaction vessel 30, and a waste tank 28.
Fig. 14 is an explanatory view illustrating procedures for loading the column.
Fig. 15 is a cross-sectional view to explain the construction of an alternative reaction vessel fixture 136.
Fig. 16 is an explanatory view illustrating an alternate bottom surface of the cartridge 50.
Fig. 17 is an explanatory view illustrating chambers and flow passages inside a cartridge 250.
Fig. 18 is an explanatory view illustrating the positions of porous materials inside the cartridge 250.
Fig. 19 is a diagram to explain procedures for amplifying DNA in the reaction apparatus 90.
Fig. 20 is a diagram to explain procedures for fragmentizing DNA in the reaction apparatus 90.
Fig. 21 is a diagram to explain procedures for labeling DNA in the reaction apparatus 90.
Fig. 22 is an explanatory view illustrating a flow route when a DNA solution is caused to flow toward a column portion 206.
Fig. 23 is an explanatory view illustrating a flow route when an elution buffer is sucked out through the column portion 206.
Fig. 24 is an external view of a cartridge 350.
Fig. 25 is an explanatory view illustrating a first layer 351a of the cartridge 350.
Fig. 26 is an explanatory view illustrating a second layer 351b of the cartridge 350.
Fig. 27 is an explanatory view illustrating a third layer 351c of the cartridge 350.
Fig. 28 is an explanatory view illustrating a fourth layer 351d of the cartridge 350.
Fig. 29 is a plan view and a front view of a mini-array 350b.
Fig. 30 is a partial cross-sectional view, taken along B-B', of the cartridge 350.
Fig. 31 is a diagram to explain procedures for amplifying and regulating genome DNA of rice.
Fig. 32 is a diagram to explain procedures for reacting the regulated DNA with DNA spots.
Fig. 33 is an external view of a cartridge 450.
Fig. 34 is an explanatory view illustrating a first layer 451a of a cartridge body 450a.
Fig. 35 is an explanatory view illustrating a second layer 451b of the cartridge body 450a.
Fig. 36 is an explanatory view illustrating a third layer 451c of the cartridge body 450a.
Fig. 37 is a diagram to explain procedures for post-treatment of a CGH array.

Best Modes for Carrying Out the Invention

[First Embodiment]

Best modes for carrying out the present invention will be described below with reference to the drawings. Fig. 1 is an overall view illustrating general construction of a reaction apparatus 90, which is one embodiment practicing the present invention. Fig. 2 is an explanatory view illustrating a cartridge mounting mechanism 80. More specifically, Fig. 2A is an exploded perspective view, Fig. 2B is a perspective view, and Fig. 2C a cross-sectional view taken along A-A' in Fig. 2B. Fig. 3 is a cross-sectional view of a reaction vessel 30. Fig. 4 is an explanatory view illustrating layout of liquid container portions for containing liquids, flow passages for liquids and gases, etc., which are formed in a first layer 50a and a second layer 50b of a cartridge 50. Fig. 5 is an explanatory view including cross-sections of a micro check valve 160. Fig. 6 is an explanatory view illustrating the position of a porous material disposed inside the cartridge 50. Fig. 7 is an explanatory view illustrating a heat-insulated structure of the reaction apparatus 90. The cartridge 50 includes, though described in detail later, communicating ports 1a-16a and 18a each connectable to the reaction vessel 30, and an introducing port 17a. For the sake of convenience in explanation, those ports are collectively referred to as "ports". Also, liquid container portions 1-15, 17 and 18 and a waste tank 28, each capable of containing a liquid, are collectively referred to as "chambers". This embodiment is described in connection with the case where the reaction apparatus 90 is used to obtain labeled cDNA for detecting representation (abundance) of mRNA.
The reaction apparatus 90 according to this embodiment includes, as illustrated in Fig. 1, a cartridge mounting mechanism 80 capable of mounting the cartridge 50, which contains plural kinds of reagents (liquids) in individual liquid container portions in an optionally extractable manner and which has a guide portion 52 disposed on a top surface thereof, a reaction vessel 30 capable of being selectively connected to plural ports disposed in the mounted cartridge 50 and capable of containing the liquid supplied from the connected port, a rotation mechanism 32 for rotationally moving the cartridge 50 about its central axis such that any one of the ports of the cartridge 50 comes to a position where it is connected to the reaction vessel 30, a pump 34 capable of supplying the liquid contained in the liquid container portion of the cartridge 50 to the reaction vessel 30 by causing differential pressure to act upon the relevant liquid container portion, and capable of transferring the liquid(s) contained in the reaction vessel 30 to the cartridge 50, a reaction vessel fixture 36 for fixing the reaction vessel 30 to a support member 92, a Peltier device 38a for the cartridge, which can adjust temperature of the cartridge 50 mounted by the cartridge mounting mechanism 80, a Peltier device 36a for the reaction vessel, which can adjust temperature of the reaction vessel 30, a start button (not shown) through which a user instructs the start of treatment in the reaction apparatus 90, and a controller 40 for controlling the entirety of the reaction apparatus 90. The reaction apparatus 90 includes a rectangular base 90a arranged at its lowermost portion, and the support member 92 disposed in a front portion of the base 90a and having an L-shape in a side view. The support member 92 has an intermediate step surface 92a and a vertical wall portion 92b formed to extend upward on the rear side of the intermediate step surface 92a. Further, the pump 34 and the controller 40 are disposed behind the support member 92.
The cartridge mounting mechanism 80 includes, as illustrated in Fig. 2, a rotary disk 82 to which the reaction vessel 30 is inserted and which is in turn fitted to the guide portion 52 of the cartridge 50, retainers 84 for biasing the rotary disk 82 downward from above, and a rotary stage 38 (see Fig. 1) on which the cartridge 50 is placed. The rotary disk 82 is made of a fluorine-based material (e.g., Teflon (registered trade name, this is equally applied to Teflon appearing below)) in consideration of water repellency and oil repellency. The retainers 84 are made of a fluorine-based material (e.g., Teflon) in consideration of heat resistance, heat insulation, easy sliding of the rotary disk, etc. As illustrated in the A-A' cross-sectional view of Fig. 2C, the rotary disk 82 is brought into contact with a contact surface 52a, which includes a plurality of ports disposed along a circular pattern and which is positioned on the inner peripheral side of the guide portion 52 of the cartridge 50, through O-rings 54a disposed at the ports. The rotary disk 82 has one flow path 82a for causing one of the ports to be communicated with the reaction vessel 30. The O-rings 54a are disposed at the ports in one-to-one correspondence relation. The rotary disk 82 is rotatably fitted to the guide portion 52 of the cartridge 50 and makes one of the ports (i.e., any one chamber) communicated with the reaction vessel 30 through the flow path 82a in the rotary disk 82. Further, when the rotary disk 82 is in a state fitted to the guide portion 52, it blocks off communication between the other ports, which are not communicated with the flow path 82a, and the atmosphere such that the liquid can be transferred only through the port communicating with the flow path 82a. The retainers 84 serve as members for biasing the rotary disk 82 downward from above in such a manner that the cartridge 50 is kept rotatable in a state where the rotary disk 82 is held in contact with the contact surface 52a of the cartridge 50 placed on the rotary stage 38. The retainers 84 bias the rotary disk 82 downward from above while pressing two stepped portions 82b formed on an upper surface of the rotary disk 82 in opposite sides thereof, thus limiting vertical movement and rotational movement of the cartridge 50. Therefore, even when the cartridge 50 is rotationally moved, the position of the flow path 82a of the rotary disk 82 is maintained the same. As a result, only any one of the ports can be brought into a state communicating with the reaction vessel 30 by rotating the cartridge 50. A gap is formed between the rotary disk 82 and the contact surface 52a of the cartridge 50 with the O-rings 54a interposed between them. Through the gap, the atmosphere can be taken into the cartridge 50 through a first vent hole 21a (described later), and gas inside the cartridge 50 can be discharged to the outside.
The rotation mechanism 32 includes, as illustrated in Fig. 1, the rotary stage 38 on which the cartridge 50 is placed, and a motor 37 for rotationally moving the rotary stage 38 in a stepwise manner such that the rotary stage 38 is fixedly held at a predetermined connecting position. The rotary stage 38 is in the form of a disk and is rotatably journal-supported to the intermediate step surface 92a of the support member 92. The rotary stage 38 is made by electroless nickel plating of copper and has a plurality of projections (not shown) formed on its upper surface at irregular positions. The cartridge 50 has a plurality of recesses, which are formed in its bottom surface to be engageable respectively with the projections of the rotary stage 38 and which are positioned corresponding to the arrangement of the projections. The cartridge 50 can be fixedly held at a predetermined initial connecting position by placing the cartridge 50 on the rotary stage 38 while the projections on the latter and the recesses in the former are engaged with each other. The rotary stage 38 includes therein the Peltier device 38a for the cartridge, which can adjust the temperature of the cartridge 50. The cartridge 50 placed on the rotary stage 38 can be adjusted to a constant temperature by adjusting the temperature of the rotary stage 38 with the Peltier device 38a for the cartridge. The rotary stage 38 may be made of a material obtained by alimiting aluminum.
The reaction vessel fixture 36 is made by electroless nickel plating of copper and is fixed to a center of the vertical wall portion 92b of the support member 92. The reaction vessel fixture 36 serves to fixedly hold the reaction vessel 30 in a detachable manner above the cartridge 50 placed on the rotary stage 38. The reaction vessel fixture 36 includes therein the Peltier device 36a for the reaction vessel, which can adjust the temperature of the reaction vessel 30. The reaction vessel 30 can be adjusted to a constant temperature by adjusting the temperature of the reaction vessel fixture 36 with the Peltier device 36a for the reaction vessel. The reaction vessel fixture 36 may be made of a material obtained by alimiting aluminum.
The reaction vessel 30 is a made of polypropylene and has the shape of a tube tapered toward the lower side, i.e., toward its port, as illustrated in Figs. 1 and 2. The rotary disk 82 is mounted to a lower end of the reaction vessel 30 through an O-ring 54b (see Fig. 2C), and a delivery/discharge tube 34a is connected to an upper end of the reaction vessel 30 as illustrated in Fig. 1. Pressure generated with operation of the pump 34 is exerted on the reaction vessel 30 through the delivery/discharge tube 34a such that the pressure acts upon any one chamber in the cartridge 50 connected to the reaction vessel 30 through the rotary disk 82. Further, the reaction vessel 30 is used to contain the liquids selectively sucked from the liquid container portions 1-15, 17 and 18, to bubble the contained liquids, and to develop various reactions by the contained liquids. Moreover, as illustrated in Fig. 3, degassing grooves 30a are formed as vertically extending grooves in the reaction vessel 30.
The pump 34 is the so-called diaphragm pump for transporting air with change in the volume of a diaphragm and causing pressure to act upon a target connected to the pump 34. As illustrated in Fig. 1, the pump 34 is connected to the delivery/discharge tube 34a such that pressure acts upon the liquid contained in the chamber of the cartridge 50 through the delivery/discharge tube 34a and the reaction vessel 30. An intake port and a delivery port of the pump 34 can be selectively connected to the delivery/discharge tube 34a. By connecting the intake port of the pump 34 and the delivery/discharge tube 34a to each other, air pressure in the reaction vessel 30 is reduced to be able to suck the liquid from one of the liquid container portions 1-15, 17 and 18 into the reaction vessel 30. Also, by connecting the delivery port of the pump 34 and the delivery/discharge tube 34a to each other, air pressure in the reaction vessel 30 is increased to be able to push the liquid from the reaction vessel 30 into the waste tank 28.
The controller 40 is constituted as a microprocessor primarily comprising a CPU 42, and it includes a flash ROM 43 for storing various processing programs and a RAM 44 for temporarily storing or saving data. The controller 40 outputs a control signal supplied to the pump 34, including a signal instructing change of the connection state between the pump 34 and the delivery/discharge tube 34a, a control signal for the motor 37, voltages supplied to the Peltier device 36a for the reaction vessel and the Peltier device 38a for the cartridge, etc.
The cartridge 50 is a member made of a cycloolefin copolymer and, as illustrated in Fig. 2, it includes a first layer 50a formed in the shape of a circular disk, a second layer 50b also formed in the shape of a circular disk, and the guide portion 52 formed on an upper surface of the first layer 50a and used to rotate the cartridge 50 about its central axis. Each of the first layer 50a and the second layer 50b has a plurality of chambers. As illustrated in Fig. 4, the cartridge 50 includes a plurality of liquid container portions 1-15, 17 and 18 capable of containing liquids in predetermined volumes that are determined depending on the liquids to be contained, communicating ports 1a-16a and 18a disposed at respective predetermined connecting positions where any one of those chambers is communicated with the reaction vessel 30 when the cartridge 50 is rotationally moved in a selective manner, an atmosphere flowing portion 21 for communicating the liquid container portions 1-15, 17 and 18 with the atmosphere to take the atmosphere into the chambers or to discharge gases from the chambers, an introducing port 17a for introducing the liquid to the liquid container portion 17, a waste tank 28 capable of containing a liquid waste transferred from the reaction vessel 30, an outflow restriction valve 16e disposed between the waste tank 28 and the communicating port 16a to allow flow of the liquid waste from the reaction vessel 30 to the waste tank 28 and to block off flow of the liquid waste from the waste tank 28 to the reaction vessel 30, a column portion 16 capable of adsorbing a product produced by a reaction developed in the reaction vessel 30, and an inflow restriction valve 16d disposed between the liquid container portion 17 and the column portion 16 to allow flow of the liquid from the liquid container portion 17 to the column portion 16 and to block off flow of the liquid from the column portion 16 to the liquid container portion 17.
The liquid container portions 1-15, 17 and 18 are each a space formed in the shape of a tube having narrower opposite ends with liquid flowing portions on the opposite sides formed to be narrower than a liquid containing portion at the middle. As seen from comparing the liquid container portion 1 and the liquid container portion 4 with each other, for example, the liquid container portions 1-15, 17 and 18 are formed in the shapes of longer tubes as they contain larger amounts of liquids. Also, the liquid container portions 12-15 and 17 are formed in the shape of a zigzag tube with a zigzag width gradually increasing from the inner peripheral side toward the outer peripheral side of the cartridge 50 having the shape of a circular disk. Each zigzag tube is formed to become narrower as it comes closer to its port. Further, the second layer 50b positioned away from the contact surface 52a of the first layer 50a, in which the ports are formed, has the liquid container portions 14 and 15 formed in larger volumes to be able to contain larger amounts of liquids than those contained by the other chambers formed in the first layer 50a. Although the liquid container portions 1-13, 17 and 18 are communicated with each other through an atmosphere flowing passage 21d, the liquids contained in the liquid container portions 1-13, 17 and 18 are prevented from mixing with each other through the atmosphere flowing passage 21d because hydrophobic porous materials are disposed therein as described below.
The atmosphere flowing portion 21 includes the atmosphere flowing passage 21d connected to respective one ends of the liquid container portions 1-13, 17 and 18 in the first layer 50a on the outer peripheral side thereof, an atmosphere flowing passage 21e connected to respective one ends of the liquid container portions 14 and 15 in the second layer 50b on the outer peripheral side thereof, a first vent hole 21a for communicating the atmosphere flowing passages 21d and 21e with the atmosphere, and a hydrophobic porous material disposed in each of the atmosphere flowing passages 21d and 21e. In Fig. 6, the atmosphere flowing portion 21 is indicated by double hatching. The hydrophobic porous material allows passing of a liquid, but does not allow passing of the atmosphere. For example, a Teflon porous material (made by Nitto Denko Corporation, TEMISH) is used as the hydrophobic porous material. The atmosphere flowing passage 21e is connected at one end to the liquid container portions 14 and 15 and at the other end to the first vent hole 21a through a vertical flow passage which is formed to extend from a position 22c of the flow passage in the second layer 50b to a position 22b of the flow passage in the first layer 50a, whereby the atmosphere flowing passage 21e is communicated with the atmosphere through the first vent hole 21a.
The communicating ports 1a-16a and 18a are holes which are communicated respectively with the liquid container portions 1-15, 17 and 18, which are used to supply the liquids from the liquid container portions 1-15, 17 and 18, and which are formed in the upper surface of the first layer 50a on the inner peripheral side thereof (i.e., in the contact surface 52a). The communicating ports 1a-16a and 18a are disposed on the same plane (flat surface) in a circular pattern coaxial with an axis of rotation about which the cartridge 50 is rotated by the rotation mechanism 32, i.e., with the central axis of the cartridge 50. Further, the liquids contained in the liquid container portions 1-15, 17 and 18 can be supplied to the reaction vessel 30 by differential pressure acting upon the liquids contained in the liquid container portions 1-15, 17 and 18 which are connected respectively to those communicating ports. The communicating ports 14a and 15a are connected respectively to connecting positions 14c and 15c, which correspond to respective one ends of the liquid container portions 14 and 15 in the second layer 50b, through flow passages formed to vertically extend for connecting the first layer 50a and the second layer to each other.
The introducing port 17a is a hole which is communicated with the liquid container portion 17 and which is used to introduce the liquid transferred from the outside to the liquid container portion 17. The introducing port 17a is formed in the upper surface of the first layer 50a on the inner peripheral side thereof (i.e., in the contact surface 52a). The communicating port 17a is disposed on the same circular pattern as the communicating ports 1a-16a and 18a. The liquid container portion 17 is connected to the inflow restriction valve 16d through a flow passage which is formed to vertically extend from a position 24b of the flow passage at one end of the liquid container portion 17 to a position 24c in the second layer 50b. Also, the other end of the liquid container portion 17 is communicated with the atmosphere flowing passage 21d. Though described in detail later, the hydrophobic porous material not allowing passing of any liquid is disposed in the atmosphere flowing passage 21d. Thus, since a liquid cannot be introduced to the liquid container portion 17 from any end thereof, the introducing port 17a is formed such that the liquid can be introduced to the liquid container portion 17. Liquids can be introduced to the other liquid container portions 1-15 respectively from the communicating ports 1a-15a.
The column portion 16 is disposed between the communicating port 16a and the outflow restriction valve 16e, and it includes a column. It is here assumed that a ceramic column (e.g., a silica gel) is used as the column. Because the liquid contained in the liquid container portion 17 is to be transferred to the reaction vessel 30 through the communicating port 16a, the communicating port 16a communicating with the column portion 16 is communicated with the liquid container portion 17 and is disposed in the upper surface of the first layer 50a on the inner peripheral side thereof (i.e., in the contact surface 52a).
The outflow restriction valve 16e and the inflow restriction valve 16d are each the so-called check valve. For example, a micro check valve 160 illustrated in Fig. 5 can be used. The micro check valve 160 is constituted by a circular valve member 161 made of, e.g., silicone rubber and a casing 162 for fixing the valve member 161 in place. The valve member 161 has such a structure that an arc-shaped slit is formed in the valve member 16 and a flap 163 positioned inside the slit is capable of swinging about a swing axis 164. The casing 162 has a valve member gripping portion 162a for gripping the valve member 161 from both sides. The valve member gripping portion 162a has a hole which is formed on the left side of the valve member 161 as viewed on the drawing and which has a diameter smaller than that of the flap 163, and a hole which is formed on the right side of the valve member 161 and which has a diameter larger than that of the flap 163, but smaller than that of the valve member 161. Therefore, when a fluid is going to flow in a direction denoted by A in the drawing, the flap 163 is inclined in the direction A about the swing axis 164 of the valve member 161 so as to form a gap through which the fluid can flow. On the other hand, when a fluid is going to flow in a direction denoted by B in the drawing, the flap 163 is urged to incline in the direction B about the swing axis 164 of the valve member 161, but the flap 163 contacts with an end surface of the casing 162. Hence, no gap is formed and the fluid cannot flow through the valve. The micro check valve 160 is installed as the outflow restriction valve 16e in the cartridge 50 in such a state that the direction A in Fig. 5 is a direction toward a position 23c of the flow passage from the column portion 16 and the direction B is an opposite direction. Also, the micro check valve 160 is installed as the inflow restriction valve 16d in the cartridge 50 in such a state that the direction A in Fig. 5 is a direction toward the column portion 16 from the position 24c of the flow passage and the direction B is an opposite direction. The micro check valve 160 used in this embodiment is under development by Suzumori Lavatory in Department of System Engineering, Faculty of Engineering, Okayama University.
As illustrated in Fig. 4, the waste tank 28 is a space formed to extend along an entire outermost periphery of the cartridge 50 as one unitary space spreading over both the first layer 50a and the second layer 50b. Inside the waste tank 28, a water-absorbing porous material, such as a sponge, capable of absorbing the liquid waste is disposed to positively hold the liquid waste within the waste tank 28 once the liquid waste has flown into there. The water-absorbing porous material is arranged so as to entirely cover an outer periphery of the cartridge 50 (see a hatched area in Fig. 6). Further, the water tank 28 is connected to the column portion 16 through a flow passage which is formed to extend from a position 23d at an end of a flow passage connected to the waste tank 28 to a position 23b of the flow passage on the inner peripheral side thereof, and through a flow passage which is formed to vertically extend from the position 23b to the position 23c of the flow passage in the second layer 50b. In other words, the fluid having passed through the column portion 16 and the outflow restriction valve 16e is introduced to the waste tank 28. A second vent hole 28a communicating with the atmosphere is formed in the upper surface of the first layer 50a at a position corresponding to the waste tank 28.
In the reaction apparatus 90 thus constructed, liquids including, e.g., reagents used to develop predetermined reactions, are contained in desired amounts in the liquid container portions of the cartridge 50, and various treatments can be executed by rotationally moving the cartridge 50 in sequence with the motor 37 to selectively change the port-connected position, and by sequentially supplying the liquid from the connected one of the liquid container portions 1-15, 17 and 18 to the reaction vessel 30 with the pump 34 to progress a predetermined reaction in the reaction vessel 30, or by transferring the liquids after the reaction to the waster tank 28 from the reaction vessel 30. Particularly, when a reaction product is to be purified, the reaction product is adsorbed on the column while the useless liquid is introduced to the waste tank 28, and a liquid is supplied to the reaction vessel 30 from the liquid container portion 17 through the column. Further, in the reaction apparatus 90, as illustrated in Fig. 7, the reaction vessel 30 is disposed outside the cartridge 50. Therefore, temperature change of the reaction vessel 30 is less conducted to the cartridge 50, and the reaction vessel 30 and the cartridge 50 can be held at different temperatures (e.g., a reaction temperature and a temperature suitable for preservation, respectively).
The operation of the reaction apparatus 90, in particular, the operation for obtaining labeled cDNA from mRNA as a sample will be described below. Fig. 8 is a diagram to explain procedures for obtaining labeled cDNA from mRNA. Fig. 8 schematically illustrates the liquid container portions 1-15 and 17 and the waste tank 28 of the cartridge 50, the communicating ports connected, and the reaction vessel 30. In Fig. 8, regarding the liquid container portions 1-15 and 17 and the waste tank 28, the types and the amounts of liquids contained therein and corresponding numerals illustrated in Fig. 4 are denoted. A blank chamber represents that no liquid is contained therein. Regarding the reaction vessel 30, an oblong circle represents that a liquid is contained in the reaction vessel 30, and a rectangle represents that treatment is executed on the contained liquid. A blank oblong circle represents that no liquid is contained in the reaction vessel 30. Each of arrows in Fig. 8 represents a direction in which a liquid or a gas flows. For the sake of convenience in explanation, step numbers are denoted in association with the reaction vessel 30.
First, the user puts mRNA as a sample in the reaction vessel 30, connects the reaction vessel 30 to the rotary disk 82, and fits the rotary disk 82 to the cartridge 50, thus preparing a reaction unit (see Fig. 2B). Then, the user opens a door (not shown) provided in a side of the reaction vessel fixture 36 and places the reaction unit on the rotary stage 38 while laterally sliding the reaction unit into a such state that the top of the reaction vessel 30 is communicated with the delivery/discharge tube 34a and the rotary disk 82 is biased downward by the retainers 84. At that time, because the retainers 84 are made of Teflon and are flexible, the reaction unit is mounted in a state where the plurality of recesses formed in the bottom surface of the cartridge 50 are engaged with the plurality of projections provided on the upper surface of the rotary stage 38, and where the reaction unit is biased downward by the retainers 84. Then, the user depresses the start button (not shown). Responsively, the CPU 42 in the controller 40 reads and executes a cDNA synthesizing and labeling process routine stored in the flash ROM 43. When that routine is started, the CPU 42 executes control as follows. First, the cartridge 50 is held at a predetermined temperature (e.g., 20°C) by the Peltier device 38a for the cartridge. Then, the motor 37 is driven to rotate the cartridge 50, to thereby communicate the communicating port 1a with the reaction vessel 30, and the pump 34 is operated to lower the air pressure in the reaction vessel 30, to thereby suck the liquid contained in the liquid container portion 1 into the reaction vessel 30 (step S100).
Next, the communicating port 2a is communicated with the reaction vessel 30 to suck the liquid contained in the liquid container portion 2 (S110). Subsequently, the temperature in the reaction vessel 30 is kept at 70°C by the Peltier device 36a for the reaction vessel, and the pump 34 is operated to continuously lower the air pressure in the reaction vessel 30 and to continuously take the atmosphere into the reaction vessel 30 through the atmosphere flowing portion 21, the liquid container portion 2, the communicating port 2a, and the flow path 82a, whereby the mixed liquids contained in the reaction vessel 30 are subjected to bubbling for 10 minutes to develop the reaction of a mixed solution in the reaction vessel 30 (step S120). In such a way, the liquids in the reaction vessel 30 can be subjected to bubbling by continuously taking in the atmosphere through the atmosphere flowing portion 21, one of the liquid container portions 1-15 and 17, the communicating port formed at one end of the one of the liquid container portions 1-15 and 17, and the flow path 82a. It is assumed that the term "bubbling" used in the following description also means such an operation. Further, as illustrated in Fig. 3, the degassing grooves 30a are provided in the reaction vessel 30. When the air pressure in the reaction vessel 30 is lowered and the atmosphere is caused to flow in through the connected port, a rise of a level of the contained liquid and adhesion of the contained liquid to a wall surface are prevented by gases flowing through the degassing grooves 30a, thus ensuring a more satisfactory reaction of the contained liquid. Returning to the explanation of Fig. 8, the temperature in the reaction vessel 30 is kept at 42°C and the bubbling is performed for 3 minutes to develop a reaction (step S130). Then, the communicating ports 3a-8a are successively communicated with the reaction vessel 30 and the liquids contained in the liquid container portions 3-8 are sucked out successively (steps S140 to S190). Then, the temperature in the reaction vessel 30 is kept at 42°C and the bubbling is performed for 40 minutes to develop a reaction (step S200). Then, the communicating port 9a is communicated with the reaction vessel 30 and the liquid contained in the liquid container portion 9 is sucked out (step 5210). Then, the temperature in the reaction vessel 30 is kept at 42°C and the bubbling is performed for 40 minutes to develop a reaction (step S220). Then, the communicating ports 10a-12a are successively communicated with the reaction vessel 30 and the liquids contained in the liquid container portions 10-12 are sucked out successively (steps S230 to S250). Then, the temperature in the reaction vessel 30 is kept at 65°C and the bubbling is performed for 60 minutes to develop a reaction (step S260). Then, the communicating ports 13a-14a are successively communicated with the reaction vessel 30 and the liquids (adsorption buffers) contained in the liquid container portions 13 and 14 are sucked out successively (steps S270 and S280).
Subsequently, the communicating port 16a is communicated with the reaction vessel 30 and, after changing the connected state of the pump 34, the pump 34 is operated to raise the air pressure in the reaction vessel 30, thus causing the mixed solution in the reaction vessel 30 to flow through the column portion 16 (step S290). Such a situation is described with reference to Fig. 9. Fig. 9 is an explanatory view illustrating a flow route through which the mixed solution flows. When the mixed solution flows through the column portion 16 via the communicating port 16a in the first layer 50a of the cartridge 50 as illustrated in Fig. 9A, only the labeled cDNA in a reaction mixture is adsorbed on the column inside the column portion 16. The liquid waste having passed through the column further passes through the outflow restriction valve 16e in the second layer 50b, as illustrated in Fig. 9B, and finally flows into the waste tank 28 after flowing through the vertical flow passage from the position 23c of the flow passage to the first layer 50a. At that time, although the liquid container portion 17 is also connected to the flow route through which the liquid waste flows, the liquid waste is prevented from flowing into the liquid container portion 17 by the inflow restriction valve 16d which is disposed upstream of the liquid container portion 17.
Subsequently, the communicating port 15a is communicated with the reaction vessel 30 and, after changing the connected state of the pump 34, the pump 34 is operated to lower the air pressure in the reaction vessel 30, thereby sucking out the liquid (wash buffer) contained in the liquid container portion 15 (step S300). Then, the communicating port 16a is communicated with the reaction vessel 30 and, after changing the connected state of the pump 34, the pump 34 is operated to raise the air pressure in the reaction vessel 30, thus causing the washing liquid in the reaction vessel 30 to flow through the column portion 16 for washing the column (step S310). The wash buffer after the washing is caused to flow into the waste tank 28 as in step S290. Further, after changing the connected state of the pump 34, the pump 34 is operated to lower the air pressure in the reaction vessel 30 while the communicating port 16a is kept communicated with the reaction vessel 30, whereby the liquid (elution buffer) contained in the liquid container portion 17 is caused to flow through the column portion 16, following which the elution buffer is sucked toward the reaction vessel 30 (step S320) and then introduced to the reaction vessel 30 (step S330). Such a situation is described with reference to Fig. 10. Fig. 10 is an explanatory view illustrating a flow route through which the liquid contained in the liquid container portion 17 flows. When the air pressure in the reaction vessel 30 is lowered, the air pressure in the column portion 16 and the flow route on the side where the liquid waste flows is also lowered. On that occasion, neither the liquid waste nor the gas is sucked out from the waste tank 28 with the provision of the outflow restriction valve 16e. However, the liquid contained in the liquid container portion 17 is sucked out from the liquid container portion 17 and flows through the vertical flow passage from the position 24b of the flow passage at one end of the liquid container portion 17 to the position 24c in the second layer 50b and further through the column portion 16, followed by finally entering the reaction vessel 30. At that time, the labeled cDNA adsorbed on the column is eluted into the elution buffer, thus resulting in such a state that a solution including the labeled cDNA is contained in the reaction vessel 30. Although the introducing port 17a is formed in the flow passage on the inner peripheral side of the liquid container portion 17, the introducing port 17a is closed by the rotary disk 82 so as to prevent the atmosphere from flowing in and the liquid from flowing out through the introducing port 17a. Thus, when the product adsorbed on the column is eluted and sucked out from the column, the product is eluted with the liquid contained in the liquid container portion 17 which is connected to the column through the inflow restriction valve 16d, and is then sucked into the reaction vessel 30. Further, the operation of the pump 34 is continued and the temperature in the reaction vessel 30 is kept at 80°C for 30 minutes to concentrate the mixed solution that has been sucked into the reaction vessel 30 (step S340). The labeled cDNA is thereby obtained.
As described above, the bubbling and the reaction of the liquid(s) contained in the reaction vessel 30 are progressed by connecting any one of the plural liquid container portions 1-15 and 17 to the reaction vessel 30, acting differential pressure upon the liquid in the connected liquid container portion with the operation of the pump 34 to introduce the relevant liquid into the reaction vessel 30, and by continuing the operation of the pump 34 to take the atmosphere, which flows in through the atmosphere flowing passage 21, into the reaction vessel 30. Further, the mRNA having been initially put in the reaction vessel 30 can be analyzed by using the labeled cDNA obtained through the above-described procedures. The labeled cDNA thus obtained is applied to a usual DAN chip, for example. After executing a hybridization reaction step and a washing step, fluorescence emitted from a spot on the DNA chip is captured by a scanner and the intensity of a signal is processed into a numerical value. A change in representation (abundance) is measured by setting, as a control, a signal obtained from mRNA in a normal cell, and by calculating a ratio of that signal to a signal obtained from mRNA in an inspection target cell.
Here, correspondence relations between the components in this embodiment and constituent elements in the present invention are clarified. The cartridge 50 in this embodiment corresponds to a fluid containing cartridge in the present invention, the communicating ports 1a-16a and 18a correspond to connecting and communicating portions, the liquid container portions 1-15 and 17 correspond to container portions, and a combination of the first layer 50a and the second layer 50b corresponds to a housing. Further, each of the first layer 50a and the second layer 50b corresponds to a divided layer, the waste tank 28 corresponds to a reservoir portion, the communicating port 16 corresponds to a coupling and communicating portion, the outflow restriction valve 16e corresponds to an outflow restricting portion, the inflow restriction valve 16d corresponds to an inflow restriction portion, the water-absorbing porous material corresponds to an absorbing material, the first vent hole 21a corresponds to an atmosphere hole, the hydrophobic porous material corresponds to a porous material, the cartridge mounting mechanism 80 corresponds to mounting unit, the rotation mechanism 32 corresponds to a moving unit, the pump 34 corresponds to a pressure applying unit, the rotary disk 82 corresponds to a contact member, the retainers 84 correspond to a fixing member, the flow path 82a corresponds to a through-hole, the Peltier device 38a for the cartridge corresponds to a cartridge temperature adjusting unit, and the Peltier device 36a for the reaction vessel corresponds to a reaction-vessel temperature adjusting unit.
With the reaction apparatus 90 according to this embodiment, which has been described in detail above, in a state where the liquids are contained in the plurality of liquid container portions 1-15 and 17 which are formed in the first layer 50a and the second layer 50b of the cartridge 50 and which have predetermined volumes determined depending on the liquids to be contained, and where the reaction vessel 30 is connected to any one of the plurality of communicating ports, when pressure is applied to act upon one of the liquid container portions 1-15 and 17, which is communicated with the communicating port connected to the reaction vessel 30, the atmosphere is supplied to the one of the liquid container portions 1-15 and 17 through the atmosphere flowing passage 21 communicating with the outside, and the liquid contained in the one liquid container portion is supplied to the reaction vessel 30. Further, the cartridge 50 is rotated to communicate another communicating port with the reaction vessel 30 such that plural liquids are eventually supplied to the reaction vessel 30. On that occasion, since the atmosphere supplied through the atmosphere flowing passage 21 is further supplied to the reaction vessel 30 through the communicating port connected to the reaction vessel 30 and the one of the liquid container portions 1-15 and 17, the plural liquids contained in the reaction vessel 30 are subjected to the bubbling with the inflow of the atmosphere. Accordingly, the contained liquids can be caused to sufficiently develop a reaction in the reaction vessel 30.
Since the ports are disposed in the contact surface 52a, which is formed in the first layer 50a, in the circular pattern coaxial with the central axis of the cartridge 50, it is just required to rotate the cartridge 50 when one of the communicating ports is selectively connected to the reaction vessel 30. Therefore, the connection can be selectively changed with ease, and the liquid can be easily introduced to the reaction vessel 30, thus enabling the reaction to be sufficiently developed with ease. Further, for those ones of the liquid container portions 1-15 and 17, which are each formed in the shape of a zigzag tube with a zigzag width gradually increasing from the inner peripheral side toward the outer peripheral side of the cartridge 50 having the shape of a circular disk, each port is disposed on the inner peripheral side of the circular disk-shaped cartridge 50. Therefore, the zigzag width can be gradually increased toward the outer peripheral side while effectively utilizing a space in the cartridge 50 having the shape of a circular disk. Also, since the liquid container portions 1-15 and 17 are each in the tube-like shape, the liquids contained therein can be fully supplied to the reaction vessel 30. For those ones of the liquid container portions 1-15 and 17, which are each formed in a shape gradually narrowing toward its port, the liquid contained therein can be transferred to the reaction vessel 30 in amount as possible as close to all, and hence the reaction can be more satisfactorily developed in the reaction vessel 30. For those ones of the chambers, which are each formed in the shape of a tube having narrower opposite ends with liquid flowing portions on the opposite sides formed to be narrower than a liquid containing portion at the middle, the liquid contained in such a chamber can be prevented from flowing out when the cartridge 50 is distributed or handled, for example, and the contained liquid can be caused to sufficiently develop the reaction in the reaction vessel 30 with more ease. For those ones of the chambers, which are each formed in the shape of a longer tube to contain a larger amount of liquid, the liquid can be efficiently contained in such a chamber. Moreover, since the cartridge 50 includes the first layer 50a and the second layer 50b and the chambers are formed in any one of the first layer 50a and the second layer 50b or in a state spreading over both the first layer 50a and the second layer 50b, the chambers can be formed in a larger number. Since the chambers having larger volumes are formed in the second layer 50b, which is positioned away from the contact surface 52 including the ports formed therein, so as to contain larger amounts of liquids than the chambers formed in the first layer 50a, the number of chambers decreases in the layer positioned farther away from the contact surface 52a. Therefore, the number of vertical flow passages connecting the chambers and the ports to each other can be reduced in the second layer 50b, and hence the cartridge 50 is easier to fabricate. Further, since the outflow restriction valve 16e, the column portion 16, and the inflow restriction valve 16d are provided, it is possible to, without changing the connection between the reaction vessel 30 and the communicating port 16a, adsorb the product produced with the reaction in the reaction vessel 30 on the column inside the column portion 16, to introduce the liquid in the liquid container portion 17 to the column portion 16, and then to return the introduced liquid to the reaction vessel 30. Since the water-absorbing porous material capable of absorbing the liquid waste is disposed in the waste tank 28, the liquid waste introduced to the waste tank 28 from the reaction vessel 30 can be more positively held in the waste tank 28 once the liquid waste has been introduced to there. Since the hydrophobic porous material is disposed in the atmosphere flowing portion 21, the liquids contained in those ones of the chambers, which are connected to the atmosphere flowing portion 21, can be prevented from flowing out to the outside through the atmosphere flowing portion 21. Further, for those ones of the chambers, which are connected to the atmosphere flowing passage 21, the liquid in such a chamber can be prevented from flowing out to the outside through the atmosphere flowing portion 21, and the number of first vent holes 21a to be formed can be reduced. In addition, since the reaction vessel 30 is a member having the shape of a tube tapered toward its port, all the liquid can be easily caused to flow between the chamber and the reaction vessel 30 through the port, and the liquid in the reaction vessel 30 can be easily subjected to the bubbling with the atmosphere introduced to flow through the port.
Since the CPU 42 executes the cDNA synthesizing and labeling process routine set in advance and controls the rotation mechanism 32 and the pump 34, the liquids can be transferred so as to develop the reaction in accordance with the procedures of the cDNA synthesizing and labeling process routine. Comparing with the case where the user performs operations to supply the liquids to the reaction vessel 30 and to develop the reaction at the predetermined temperature for the predetermined time, therefore, each step of the process can be more reliably executed under preset conditions and hence a variation in the reaction results can more positively suppressed. Further, since the liquids are sucked from the liquid container portions 1-15 and 17 into the reaction vessel 30 by lowering the air pressure in the reaction vessel 30 with the operation of the pump 34 and the liquid waste is pushed out from the reaction vessel 30 to the waste tank 28 by raising the air pressure in the reaction vessel 30, the liquids can be transferred with a comparatively simple arrangement for changing the pressure in the reaction vessel 30. Since the cartridge 50 is rotated about the axis of rotation to selectively connect one of the ports and the reaction vessel 30 to each other, the desired rotation can be more easily performed than the case of rotating the rotary disk 82 to which the delivery/discharge tube 34a is connected. Since the cartridge 50 is mounted in place and the reaction vessel 30 is connected to the rotary disk 82 with the aid of both the rotary disk 82 contacting with the contact surface 52a and the retainers 84 biasing the rotary disk 82 against the contract surface 52a while the cartridge 50 is kept rotatable, the cartridge 50 can be mounted in a state where the chambers and the reaction vessel 30 can be comparatively easily communicated with each other in a selective manner. In addition, with the provision of the Peltier device 38a for the cartridge and the Peltier device 36a for the reaction vessel, it is possible to separately adjust the mounted cartridge 50 to temperature not causing the contained liquids to develop the reaction and the reaction vessel 30 to temperature suitable for the reaction. As a result, the liquids contained in the cartridge 50 can be caused to sufficiently develop the reaction in the reaction vessel 30 regardless of the temperature of the mounted cartridge 50.
Be it noted that the present invention is in no way limited to the above-described first embodiment and can be carried out in various embodiments within the technical scope of the present invention.
For example, while in the above-described first embodiment the cartridge 50 and the reaction apparatus 90 are used to obtain labeled cDNA from mRNA for detecting the representation (abundance) of mRNA, the present invention may also be applied to other chemical reactions. In other words, the cartridge 50 may contain liquids adapted for other chemical reactions, and the reaction apparatus 90 may develop the other reactions by using that cartridge 50. Even in such a case, the liquids contained in the liquid container portions 1-15 and 17 can be caused to sufficiently develop the reactions in the reaction vessel 30. On that occasion, the liquid container portions 1-15 and 17 formed in the cartridge 50 may be formed to have volumes capable of containing respective amounts of liquids used in the other chemical reactions. Also, the reaction apparatus 90 may store respective process routines for the other chemical reactions and may execute the other chemical reactions in accordance with those routines. That point is similarly applied to second and third embodiment described later. For example, the cartridge may contain liquids for producing genome DNA that is labeled for detection of SNP, and the reaction apparatus may execute chemical reactions in accordance with an SNP variation process routine, i.e., a routine for producing the labeled genome DNA. Fig. 11 is an explanatory view illustrating a cartridge 150 containing the liquids used for detection of SNP, and Fig. 12 is a diagram to explain procedures for amplifying and labeling genome DNA. As illustrated in Fig. 11, the cartridge 150 is similar to the above-described cartridge 50 except that the liquid container portions 14, 15 and 17 and the column portion 16 are not provided along with the flow passages, the ports, the outflow restriction valve, the inflow restriction valve, and the waste tank 28 which are connected to the formers, and that the liquid container portions 1-13 are designed in shapes corresponding to the volumes required for the liquids to be contained there. Hence, the same components are denoted by the same characters and detailed descriptions of those components are omitted. A reaction apparatus used herein has a similar structure to that of the above-described reaction apparatus 90 except that the SNP variation process routine is stored in the flash ROM 43. Hence, the following description is made by using similar characters to those used in the reaction apparatus 90.
When using the cartridge 150 to detect SNP, the user first puts, as a sample, genome DNA in the reaction vessel 30 and places the reaction vessel 30 on the rotary stage 38 in a similar manner to that in the above-described first embodiment. Then, the user depresses the start button (not shown). Responsively, the CPU 42 in the controller 40 reads and executes the SNP variation process routine stored in the flash ROM 43. When that routine is started, the CPU 42 executes control as follows. First, the cartridge 50 is held at a predetermined temperature (e.g., 20°C) by the Peltier device 38a for the cartridge. Then, the communicating ports 1a-3a are successively communicated with the reaction vessel 30 to successively suck out the liquids contained in the liquid container portions 1-3 (steps S400 to S420). After keeping the temperature in the reaction vessel 30 at 95°C and developing a reaction under the bubbling for 15 minutes, the reaction is continued by repeating 40 cycles of the bubbling for 30 seconds with the temperature in the reaction vessel 30 kept at 95°C, the bubbling for 1 second at the temperature of 80°C, and the bubbling for 6 minutes at the temperature of 68°C (step S430). Then, the temperature in the reaction vessel 30 is kept at 10°C for 1 minute to stop the reaction (step S440). Then, the pump 34 is operated and the temperature in the reaction vessel 30 is kept at 80°C for 30 minutes to concentrate a mixed solution sucked into the reaction vessel 30 (step S450), thereby obtaining a dried solid of amplified DNA. Subsequently, the communicating ports 4a-7a are successively communicated with the reaction vessel 30 to successively suck out the liquids contained in the liquid container portions 4-7 (steps S460 to S490). Then, a reaction is developed by executing the bubbling for 5 minutes with the temperature in the reaction vessel 30 kept at 95°C, the bubbling for 1 minute at the temperature of 50°C, and the bubbling for 60 minutes at the temperature of 58°C (step S500). Then, the temperature in the reaction vessel 30 is kept at 10°C for 1 minute to stop the reaction (step S510). Then, the pump 34 is operated and the temperature in the reaction vessel 30 is kept at 80°C for 30 minutes to concentrate a mixed solution sucked into the reaction vessel 30 (step S520), thereby obtaining a dried solid of DNA having been subjected to ligation. Subsequently, the communicating ports 8a-13a are successively communicated with the reaction vessel 30 to successively suck out the liquids contained in the liquid container portions 8-13 (steps S530 to S580). After keeping the temperature in the reaction vessel 30 at 95°C and developing a reaction under the bubbling for 1 minute, the reaction is continued by repeating 25 cycles of the bubbling for 30 seconds with the temperature in the reaction vessel 30 kept at 95°C, the bubbling for 6 minutes at the temperature of 55°C, and the bubbling for 30 seconds at the temperature of 72°C (step S590). Then, the temperature in the reaction vessel 30 is kept at 10°C for 1 minute to stop the reaction (step S600). Then, the pump 34 is operated and the temperature in the reaction vessel 30 is kept at 80°C for 15 minutes to concentrate a mixed solution sucked into the reaction vessel 30 (step S610), thereby obtaining labeled DNA having been subjected to ligation. The thus-obtained labeled DNA having been subjected to ligation is utilized, for example, by applying the labeled DNA to a usual DAN chip. After executing a hybridization reaction step and a washing step, fluorescence emitted from a spot on the DNA chip is captured by a scanner and the intensity of a signal is processed into a numerical value. SNP is discriminated based on a signal ratio between two colors.
In the above-described first embodiment, even those chambers containing comparatively small amounts of liquids, e.g., the liquid container portions 1, 2, 8 and 9, are also formed on the outer peripheral side of the first layer 50a in the form of a disk. However, the chambers containing comparatively small amounts of liquids may be formed at positions closer to the inner peripheral side, while other chambers containing comparatively large amounts of liquids may be formed at positions closer to the outer peripheral side. With such an arrangement, the chambers containing larger amounts of liquids can be formed more easily. That point is similarly applied to the second and third embodiments described later.
In the above-described first embodiment, the cartridge 50 has the shape of a circular disk. However, the cartridge may have a rectangular shape, for example, other than the shape of a circular disk. That point is similarly applied to the second and third embodiments described later.
In the above-described first embodiment, the cartridge 50 is rotated to relatively move the reaction vessel 30 and the cartridge 50. However, the relative movement between the reaction vessel 30 and the cartridge 50 is not limited to the rotation so long as they can be relatively moved. For example, the reaction vessel 30 and the cartridge 50 may be linearly moved. In such a case, the cartridge 50 may be provided with a plurality of linearly arranged ports which can be selectively connected to the reaction vessel 30. That point is similarly applied to the second and third embodiments described later.
While, in the above-described first embodiment, the reaction apparatus 90 includes the Peltier device 38a for the cartridge and the Peltier device 36a for the reaction vessel, it may not include those Peltier devices 38a and 36a. Even in such a case, the liquids contained in the liquid container portions 1-15 and 17 can be caused to sufficiently develop the reactions in the reaction vessel 30. That point is similarly applied to the second and third embodiments described later.
While, in the above-described first embodiment, the cartridge 50 includes the column portion 16, it may not include the column portion 16. Also, while the cartridge 50 includes the inflow restriction valve 16d and the outflow restriction valve 16e, it may not include those valves. That point is similarly applied to the second embodiment described later.
In the above-described first embodiment, the reaction apparatus 90 is constructed to selectively connect one of the ports to the reaction vessel 30 by rotating the cartridge 50 such that the reaction vessel 30 and the cartridge 50 are relatively moved. However, one of the ports may be selectively connected to the reaction vessel 30 by rotating the reaction vessel 30 such that the reaction vessel 30 and the cartridge 50 are relatively moved. That point is similarly applied to the second and third embodiments described later.
While, in the above-described first embodiment, the ports are disposed in the circular pattern coaxial with the central axis of the cartridge 50, the axis of the circle along which the ports are disposed is not limited to the central axis of the cartridge 50. For example, when one of the ports is selectively connected to the reaction vessel 30 by rotating the cartridge 50 relative to the reaction vessel 30 with an axis of rotation which is set to an axis differing from the central axis of the cartridge 50, the different axis of rotation may be the axis of a circle along which the ports are disposed. When the reaction vessel 30 is rotationally moved relative to the cartridge 50, an axis of rotation about which the reaction vessel 30 is rotationally moved may be the axis of the circle along which the ports are disposed. That point is similarly applied to the second and third embodiments described later.
While, in the above-described first embodiment, the hydrophobic porous material is disposed in the atmosphere flowing passages 21d and 21e, it may not be disposed therein. Also, while the water-absorbing porous material is disposed in the waste tank 28, it may not be disposed therein. That point is similarly applied to the second embodiment described later.
While, in the above-described first embodiment, the used liquid flows into only the waste tank 28, it may flow into the chamber other than the waste tank 28. For example, the used liquid may flow into the liquid container portion 18 in which no liquid is contained in the cDNA synthesizing and labeling process routine described above. In such a case, a water-absorbing porous material, such as a sponge, capable of absorbing the liquid may be disposed in one or more of the chambers into which the liquid flows and from which the liquid is no more transferred once introduced to there. That point is similarly applied to the second embodiment described later.
While, in the above-described first embodiment, the liquid container portions 1-13, 17 and 18 are coupled with each other at the outer peripheral side thereof by the atmosphere flowing passage 21d, the liquid container portions 1-13, 17 and 18 may not be coupled with each other at the outer peripheral side thereof by the atmosphere flowing passage 21d. That point is similarly applied to the second and third embodiments described later.
While, in the above-described first embodiment, the pump 34 is a diaphragm pump, it may be the so-called tube pump for causing pressure to act upon a target connected to a tube by squeezing the tube with a roller. That point is similarly applied to the second embodiment described later. In such a case, an amount of air to be transported can be finely controlled by employing a stepping motor to perform the squeezing operation. In particular, by employing the tube pump provided with the stepping motor and a liquid reservoir, the elution buffer can be caused to flow through the column and to be introduced to the reaction vessel 30 again without using a check valve (see Fig. 13). Fig. 13 is an explanatory view illustrating connection relations among a column 106, the reaction vessel 30, and the waste tank 28 when the tube pump provided with the stepping motor is used as the pump 34. As illustrated in Fig. 13, the column 106 is connected to a communicating port 102 through a flow passage 104 and is also connected to the waste tank 28 through a flow passage 108, a liquid reservoir 110, and a flow passage 112. The elution buffer is contained in a liquid container portion 116 which is connected to another communicating port 114. When the liquid contained in the reaction vessel 30 is caused to flow through the column 106, the communicating port 102 and the reaction vessel 30 are communicated with each other through the rotary disk 82. Thereafter, the tube pump is operated to cause the liquid to flow through the column 106, and the liquid waste is transferred to the waste tank 28. When the elution buffer is caused to flow through the column 106, the communicating port 114 and the reaction vessel 30 are first communicated with each other through the rotary disk 82, and the tube pump is then operated to introduce the elution buffer in the liquid container portion 116 to the reaction vessel 30. Further, the communicating port 102 and the reaction vessel 30 are communicated with each other, and the tube pump is operated to cause the elution buffer to flow through the column 106 and to be accumulated in the liquid reservoir 110. In other words, the elution buffer is accumulated in the liquid reservoir 110 without being transferred to the waste tank 28 by finely controlling an amount of air to be transported by the tube pump. The tube pump is then operated to suck the elution buffer accumulated in the liquid reservoir 110 such that the elution buffer is caused to flow through the column 106 for return to the reaction vessel 30 again. Thus, after transferring the liquid waste to the waste tank 28, the elution buffer can be caused to flow through the column 106 and to be introduced to the reaction vessel 30 again without using a check valve. Because the water-absorbing porous material, such as a sponge, is filled in the waste tank 28, the liquid waste is prevented from flowing backward and contaminating the elution buffer.
While the above-described first embodiment employs the cartridge 50 and the reaction apparatus 90, a reaction unit may be constituted by the cartridge 50, the rotary disk 82, and the reaction vessel 30, or a unit-adapted reaction apparatus may be constituted by utilizing such a reaction unit. The unit-adapted reaction apparatus may be, for example, a reaction apparatus constituted by excluding the rotary disk 82 and the reaction vessel 30 from the reaction apparatus 90 described above. Even in such a case, similar advantages to those in the above-described first embodiment can also be obtained. That point is similarly applied to the second and third embodiments described later.
In the above-described first embodiment, the reaction apparatus 90 includes the controller 40 for executing the cDNA synthesizing and labeling process routine to develop the reactions of the liquids. However, the reaction apparatus 90 may include a switch for operating the pump 34 and a switch for rotating the rotary state such that the user manually performs the operations for developing a series of reactions. Even in such a case, the liquids contained in the liquid container portions 1-15 and 17 can be caused to sufficiently develop the reactions in the reaction vessel 30. That point is similarly applied to the second and third embodiments described later.
While, in the above-described first embodiment, the chambers are formed in the first layer 50a and the second layer 50b depending on the amounts of liquids to be contained, the chambers may be formed in the first layer 50a and the second layer 50b depending on the degree of necessity of temperature adjustment. More specifically, when the cartridge 50 is in a state mounted to the reaction apparatus 90, the second layer 50b is positioned closer to the rotary stage 38, which includes the Peltier device 38a for the cartridge, than the first layer 50a, the temperature adjustment can be more reliably performed on the second layer 50b than the first layer 50a. Taking into account the above point, a chamber containing a liquid having higher necessity of the temperature adjustment may be formed in the second layer 50b positioned closer to the rotary stage 38 that is subjected to the temperature adjustment. In such a case, a chamber containing a liquid having higher necessity of the temperature adjustment may be formed at a position closer to the center of the cartridge 50. The reason is that the temperature adjustment can be more easily performed at a position farther away from the atmosphere, i.e., closer to the center of the cartridge 50. On that occasion, a blank chamber may be formed on the outer peripheral side of the cartridge 50. Air in the blank chamber serves to more effectively reduce the influence of the atmosphere and to realize the temperature adjustment with more ease. The blank chamber may be a chamber dedicated for heat insulation or a waste tank to which the liquid waste is introduced.
While, in the above-described first embodiment, the cartridge 50 includes one waste tank 28, the cartridge may include a plurality of waste tanks. For example, the cartridge may include a plurality of waste tanks such that at least one of the liquid wastes generated during a series of reaction procedures using the cartridge, which is to be used again later, can be introduced to a waste tank differing from a waste tank for the other liquid wastes. In such a case, the waste tank containing the liquid waste to be used again later and the other waste tank may be connected to different communicating ports, respectively. As an alternative, the waste tank containing the liquid waste to be used again later and the waste tank containing the other liquid wastes may use a common communicating port, and a selector valve for selectively changing flow passages may be used to select the waste tank to which the liquid waste(s) is introduced.
In the above-described first embodiment, the column in the column portion 16 may be loaded in accordance with the following procedures. Fig. 14 is an explanatory view illustrating the procedures for loading the column. Fig. 14 illustrates a vertical cross-section of the column portion 16. First, the column is fitted into a tubular laterally-sealing member 62 (e.g., a Teflon tube or a heat shrinkable tube (see Fig. 14A). The laterally-sealing member 62 serves to prevent the liquid, which has flown into the column, from flowing out from the lateral surface of the column. Then, the column integral with the laterally-sealing member 62 is press-fitted to a hole which is formed in the column portion 16 so as to extend over both the first layer 50a and the second layer 50b (see Fig. 14B). Then, a disk-like cover 64 made of rubber and having an outer diameter slightly larger than the diameter of the hole in the second layer 50b is fitted to the hole from below with a gap left between the cover 64 and the bottom surface of the column (see Fig. 14C). The column portion 16 loaded with the column is thereby completed (see Fig. 14D). Thus, since the column is mounted by press-fitting, it can be loaded in a comparatively simple step.
While, in the above-described first embodiment, the reaction vessel fixture 36 is constituted as one member including the door that can be opened forward, it may be constituted by two fixing members as illustrated in Fig. 15. Fig. 15 is a cross-sectional view to explain the construction of an alternative reaction vessel fixture 136. As illustrated in Fig. 15, the reaction vessel fixture 136 comprises an upper fixing member 136a and a lower fixing member 136b. The upper fixing member 136a and the lower fixing member 136b are connected to each other through a swing axis A such that both the members are able to swing in a direction C or a direction D denoted in Fig. 15. The delivery/discharge tube 34a is connected to the upper fixing member 136a. The lower fixing member 136b has a hole to which the reaction vessel 30 is fitted, and includes the Peltier device 36a for the reaction vessel. In such a case, the cartridge 50 and the reaction vessel 30 are mounted through the following procedures. First, the cartridge 50 including the rotary disk 82 mounted thereto is installed on the rotary stage 38 while the cartridge 50 is slid from a side. Then, the upper fixing member 136a is opened in the direction C and the reaction vessel 30 is fitted to the lower fixing member 136b. Thereafter, the upper fixing member 136a is closed in the direction D. After closing the upper fixing member 136a in the direction D, the upper fixing member 136a is fixed in a state being pressed against the lower fixing member 136b by using a hook (not shown). As a result, the reaction vessel 30 is sealed off at the upper and lower sides by O- rings 136c and 54b, respectively. Thus, both the upper and lower sides of the reaction vessel 30 can be sealed off by the O- rings 136c and 54b by one operation of closing the upper fixing member 136.
In the above-described first embodiment, as illustrated in Fig. 16, the recesses formed in the bottom surface of the cartridge 50 to control the rotational position thereof may be formed as a T-shaped groove made up of a groove 251 extending diametrically and a groove 252 extending in the radial direction perpendicularly to the groove 251. In such a case, corresponding to the shape of the bottom surface of the cartridge, a T-shaped land made up of a land extending diametrically and a land extending in the radial direction perpendicularly to the former is formed on the upper surface of the rotary stage 38.
In the above-described first embodiment, when the reaction vessel 30 is connected to any one of the communicating ports 1a-16a and 18a by rotating the rotary stage 38 in the backward direction with the motor 37, such an operation may be performed by first rotating the rotary stage 38 in the backward direction to a position beyond the communicating port to be connected, and then rotating the rotary stage 38 in the forward direction to the position of the communicating port to be connected. For example, prior to executing the process of step S300, the rotary stage 38 is first rotated in the reversed direction to a position closer to the communicating port 14a after passing the communicating port 15a to be connected. Then, the rotary stage 38 rotated in the forward direction to a position where the communicating port 15a and the reaction vessel 30 are opposed to each other. Such an operation contributes to suppressing deterioration of accuracy in control of the rotational position of the rotary stage 38, which is caused by backlash of a gearing associated with the motor 37.
In the above-described first embodiment, as illustrated in Fig. 15, a vent valve 134 capable of communicating the delivery/discharge tube 34a with the atmosphere may be disposed in the delivery/discharge tube 34a. In such a case, when the pump 34 is operated, the pump 34 starts to be operated with the vent valve 134 being open, to thereby eliminate the backlash of the gearing associated with the motor for driving the pump 34. Thereafter, the vent valve 134 is closed and the pump 34 is operated to deliver or discharge air in a desired amount. With such an operation, air can be delivered or discharged in a manner not generating an error that is caused due to the backlash of the gearing associated with the motor for driving the pump 34.
In the above-described first embodiment, as illustrated in Fig. 15, the vent valve 134 capable of communicating the delivery/discharge tube 34a with the atmosphere may be disposed in the delivery/discharge tube 34a, and the vent valve 134 may be opened for a predetermined time after introducing the liquid sucked from the cartridge 50 to the reaction vessel 30. With such an operation, the liquid having been sucked into the reaction vessel 30 and raised to an upper level in the reaction vessel 30 can be dropped to the bottom of the reaction vessel 30.
In the above-described first embodiment, when the liquids are caused to develop the reaction in the reaction vessel 30, the reaction is developed under substantially normal (atmospheric) pressure by continuously taking in the atmosphere to the reaction vessel 30. However, the reaction may be developed under pressurization or depressurization. For example, the reaction may be developed in a pressurized state or a depressurized state that is created in the reaction vessel 30 by forming a communicating port having no hole in the upper surface of the cartridge 50, rotating the cartridge 50 to make the reaction vessel 30 opposed to the communicating port when the reaction is developed under pressurization or depressurization, and then operating the pump 34. In the case of depressurization, the liquid in the reaction vessel 30 can be more quickly concentrated by heating the interior of the reaction vessel 30 with the Peltier device 36a for the reaction vessel. In the case of pressurization, a pressurization reaction can be developed.
In the above-described first embodiment, as illustrated in Fig. 15, a motor 72 capable of rotating a magnet 70 may be disposed at a side of the reaction vessel fixture 36 and a rotor 74 including a magnet may be put in the reaction vessel 30 such that the rotor 74 is rotated by rotating the magnet 70 with the motor 72. With such an arrangement, the liquids in the reaction vessel 30 can be stirred to develop the reaction. A neodymium magnet may be used as each of the magnet included in the rotor 74 and the magnet 70. Also, with the arrangement illustrated in Fig. 15, since the rotor 74 is positioned above the magnet 70, the rotor 74 is rotated in an inclined state with the rotation of the magnet 70. Further, since the magnet 70 arranged below the rotor 74 attracts the rotor 74 downward, the rotor 74 can be prevented from floating up by the action of surface tension of the liquid contained in the reaction vessel 30. Even when the rotor 74 is floated up, the liquid in the reaction vessel 30 can be stirred with the rotation of the rotor 74.

[Second Embodiment]

Fig. 17 is an explanatory view illustrating layout of liquid container portions for containing liquids, flow passages for liquids and gases, etc. which are provided in a first layer 250a and a second layer 250b of a cartridge 250 according to a second embodiment. Fig. 18 is an explanatory view illustrating the positions of porous materials disposed inside the cartridge 250. A reaction apparatus according to this embodiment has a similar structure to that of the reaction apparatus 90 according to the first embodiment except that the cartridge 250 is used instead of the cartridge 50, and that a DNA amplifying process routine, a DNA fragmentizing process routine, and a DNA labeling process routine, which are executed by using the cartridge 250, are stored in the flash ROM 43. In the following, therefore, the reaction apparatus according to this embodiment is described as the reaction apparatus 90. The same components are denoted by the same characters and detailed descriptions of those components are omitted. The cartridge 250 includes, though described in detail later, communicating ports 201a-206a, 208a-211a, 213a-217a, 219a and 220a each connectable to the reaction vessel 30, and introducing ports 207a, 212a and 218a. For the sake of convenience in explanation, those ports are collectively referred to as "ports". Also, liquid container portions 201-205, 207-210, 212-216 and 218-220 and a waste tank 228, each capable of containing a liquid, are collectively referred to as "chambers". This embodiment is described in connection with the case where the reaction apparatus 90 is used to amplify DNA.
The cartridge 250 is a member made of a cycloolefin copolymer and, as illustrated in Fig. 17, it includes a first layer 250a and a second layer 250b each formed in the shape of a circular disk, and a guide portion (not shown) which is similar to the above-described guide portion 52 and which is formed on an upper surface of the first layer to be used to rotate the cartridge 250 about its central axis. Each of the first layer 250a and the second layer 250b has a plurality of chambers. As illustrated in Fig. 17, the cartridge 250 includes a plurality of liquid container portions 201-205, 207-210, 212-216 and 218-220 capable of containing liquids in predetermined volumes that are determined depending on the liquids to be contained, communicating ports 201a-206a, 208a-211a, 213a-217a, 219a and 220a disposed at respective predetermined connecting positions where any one of the liquid container portions 201-205, 207-210, 212-216 and 218-220 is communicated with the reaction vessel 30 when the cartridge 250 is rotationally moved in a selective manner, an atmosphere flowing portion 221 for communicating the liquid container portions 201-205, 207-210, 212-216 and 218-220 with the atmosphere to take the atmosphere into the liquid container portions 201-205, 207-210, 212-216 and 218-220 or to discharge gases from the liquid container portions 201-205, 207-210, 212-216 and 218-220, introducing ports 207a, 212a and 218a for introducing the liquids to the liquid container portions 207, 212 and 218, a waste tank 228 capable of containing a liquid waste transferred from the reaction vessel 30, outflow restriction valves 206e, 211e and 217e disposed respectively between the waste tank 228 and the communicating ports 206a, 211a and 217a to allow flow of the liquid waste from the reaction vessel 30 to the waste tank 228 and to block off flow of the liquid waste from the waste tank 228 to the reaction vessel 30, column portions 206, 211 and 217 capable of adsorbing products produced by reactions developed in the reaction vessel 30, and inflow restriction valves 206d, 211d and 217d disposed respectively between the liquid container portions 207, 212 and 218 and the column portions 206, 211 and 217 to allow flow of the liquids from the liquid container portions 207, 212 and 218 to the column portions 206, 211 and 217 and to block off flow of the liquids from the column portions 206, 211 and 217 to the liquid container portions 207, 212 and 218.
The liquid container portions 201-205, 207-210, 212-216 and 218-220 are each a space formed in the shape of a tube having narrower opposite ends with liquid flowing portions on the opposite sides formed to be narrower than a liquid containing portion at the middle. As seen from comparing the liquid container portion 201 and the liquid container portion 204 with each other, for example, the liquid container portions 201-205, 207-210, 212-216 and 218-220 are formed in the shapes of longer tubes as they contain larger amounts of liquids. Also, the liquid container portions 202-205, 207-210, 212, 213, 215, 218 and 219 are formed in the shape of a zigzag tube with a zigzag width gradually increasing from the inner peripheral side toward the outer peripheral side of the cartridge 250 having the shape of a circular disk. Each zigzag tube is formed to become narrower as it comes closer to its port. Further, the second layer 250b positioned away from a contact surface (not shown), similar to the above-described contact surface 52a, in which the ports are formed and which is positioned on the inner peripheral side of a guide portion of the first layer 250a, has the liquid container portions 204, 205 and 216 formed in larger volumes to be able to contain larger amounts of liquids than those contained by the other chambers formed in the first layer 250a. Although the liquid container portions 201-203, 207-210, 212-215 and 218-220 are communicated with each other through an atmosphere flowing passage 221d, the liquids contained in the liquid container portions 201-203, 207-210, 212-215 and 218-220 are prevented from mixing with each other through the atmosphere flowing passage 221d because hydrophobic porous materials are disposed therein as described below.
The atmosphere flowing portion 221 includes the atmosphere flowing passage 221d connected to respective one ends of the liquid container portions 201-203, 207-210, 212-215 and 218-220 in the first layer 250a on the outer peripheral side thereof, an atmosphere flowing passage 221e connected to respective one ends of the liquid container portions 204, 205 and 216 in the second layer 250b on the outer peripheral side thereof, a first vent hole 221a for communicating the atmosphere flowing passages 221d and 221e with the atmosphere, and a hydrophobic porous material disposed in each of the atmosphere flowing passages 221d and 221e. In Fig. 18, the atmosphere flowing portion 221 is indicated by double hatching. The hydrophobic porous material allows passing of a liquid, but does not allow passing of the atmosphere. For example, a Teflon porous material (made by Nitto Denko Corporation, TEMISH) is used as the hydrophobic porous material. The atmosphere flowing passage 221e is connected at one end to the liquid container portions 204, 205 and 216 and at the other end to the first vent hole 221a through vertical flow passages which are formed to extend from positions 222c and 223c of the flow passages in the second layer 250b to positions 222b and 223b of the flow passages in the first layer 250a, whereby the atmosphere flowing passage 21e is communicated with the atmosphere through the first vent hole 221a.
The communicating ports 201a-206a, 208a-211a, 213a-217a, 219a and 220a are holes which are communicated respectively with the liquid container portions 201-205, 207-210, 212-216 and 218-220, which are used to supply the liquids from the liquid container portions 201-205, 207-210, 212-216 and 218-220, and which are formed in the upper surface of the first layer 250a on the inner peripheral side thereof (i.e., in the contact surface thereof). The communicating ports 201a-206a, 208a-211a, 213a-217a, 219a and 220a are disposed on the same plane (flat surface) in a circular pattern coaxial with an axis of rotation about which the cartridge 250 is rotated by the rotation mechanism 32, i.e., with the central axis of the cartridge 250. Further, the liquids contained in the liquid container portions 201-205, 207-210, 212-216 and 218-220 can be supplied to the reaction vessel 30 by differential pressure acting upon the liquids contained in the liquid container portions 201-205, 207-210, 212-216 and 218-220 which are connected to the associated communicating ports. The communicating ports 204a, 205a and 216a are connected respectively to connecting positions 204c, 205c and 216c, which correspond to respective one ends of the liquid container portions 204, 205 and 216 in the second layer 250b, through flow passages formed to vertically extend for connecting the first layer 250a and the second layer 250b to each other.
The introducing ports 207a, 212a and 218a are holes which are communicated respectively with the liquid container portions 207, 212 and 218, which are used to introduce the liquid transferred from the outside to the corresponding liquid container portions, and which are formed in the upper surface of the first layer 250a on the inner peripheral side thereof (i.e., in the contact surface thereof). The communicating ports 207a, 212a and 218a are disposed on the same circular pattern as the communicating ports 201a-206a, 208a-211a, 213a-217a, 219a and 220a. The liquid container portions 207, 212 and 218 are connected respectively to the inflow restriction valves 206d, 211d and 217d through flow passages which are formed to vertically extend from positions 224b-226b of the flow passages at respective one ends of the liquid container portions 207, 212 and 218 to positions 224c-226c in the second layer 250b. Also, the respective other ends of the liquid container portions 207, 212 and 218 are communicated with the atmosphere flowing passage 221d. Though described in detail later, the hydrophobic porous material not allowing passing of any liquid is disposed in the atmosphere flowing passage 221d. Thus, since liquids cannot be introduced to the liquid container portions 207, 212 and 218 from any ends thereof, the introducing ports 207a, 212a and 218a are formed such that the liquids can be introduced to the liquid container portions 207, 212 and 218. Liquids can be introduced to the other liquid container portions 201-205, 208-210, 213-216, 219 and 220 respectively from the communicating ports 201a-205a, 208a-210a, 213a-216a, 219a and 220a.
The column portions 206, 211 and 217 are disposed between the communicating ports 206a, 211a and 217a and the outflow restriction valves 206e, 211e and 217e, respectively, and include columns. It is here assumed that a ceramic column (e.g., a silica gel) is used as the column. Because the liquids contained in the liquid container portions 207, 212 and 218 are to be transferred to the reaction vessel 30 through the communicating ports 206a, 211a and 217a, respectively, the communicating ports 206a, 211a and 217a communicating with the column portions 206, 211 and 217 are communicated with the liquid container portions 207, 212 and 218, respectively, and are disposed in the upper surface of the first layer 250a on the inner peripheral side thereof (i.e., in the contact surface thereof).
The outflow restriction valves 206e, 211e and 217e and the inflow restriction valves 206d, 211d and 217d are each the so-called check valve. For example, a micro check valve 160 illustrated in Fig. 5 can be used. The micro check valve 160 is constituted by a circular valve member 161 made of, e.g., silicone rubber and a casing 162 for fixing the valve member 161 in place. The valve member 161 has such a structure that an arc-shaped slit is formed in the valve member 16 and a flap 163 positioned inside the slit is capable of swinging about a swing axis 164. The casing 162 has a valve member gripping portion 162a for gripping the valve member 161 from both sides. The valve member gripping portion 162a has a hole which is formed on the left side of the valve member 161 as viewed on the drawing and which has a diameter smaller than that of the flap 163, and a hole which is formed on the right side of the valve member 161 and which has a diameter larger than that of the flap 163, but smaller than that of the valve member 161. Therefore, when a fluid is going to flow in a direction denoted by A in the drawing, the flap 163 is inclined in the direction A about the swing axis 164 of the valve member 161 so as to form a gap through which the fluid can flow. On the other hand, when a fluid is going to flow in a direction denoted by B in the drawing, the flap 163 is urged to incline in the direction B about the swing axis 164 of the valve member 161, but the flap 163 contacts with an end surface of the casing 162. Hence, no gap is formed and the fluid cannot flow through the valve. The micro check valve 160 is installed as the outflow restriction valve 206e in the cartridge 250 in such a state that the direction A in Fig. 5 is a direction toward a position 227c of the flow passage from the column portion 206 and the direction B is an opposite direction. Also, the micro check valve 160 is installed as the inflow restriction valve 206d in the cartridge 250 in such a state that the direction A in Fig. 5 is a direction toward the column portion 206 from the position 224c of the flow passage and the direction B is an opposite direction. The inflow restriction valves 211d and 217d and the outflow restriction valves 211e and 217e are also similarly arranged with respect to the column portions 211 and 217, respectively. The micro check valve 160 used in this embodiment is under development by Suzumori Lavatory in Department of System Engineering, Faculty of Engineering, Okayama University.
As illustrated in Fig. 17, the waste tank 228 is a space formed to extend along an entire outermost periphery of the cartridge 250 as one unitary space spreading over both the first layer 250a and the second layer 250b. Inside the waste tank 228, a water-absorbing porous material, such as a sponge, capable of absorbing the liquid waste is disposed to positively hold the liquid waste within the waste tank 228 once the liquid waste has flown into there. The water-absorbing porous material is arranged so as to entirely cover an outer periphery of the cartridge 250 (see a hatched area in Fig. 18). Further, the water tank 228 is connected to the column portions 206, 211 and 217 through a flow passage which is formed to extend from a position 227d at an end of a flow passage connected to the waste tank 228 to a position 227b of the flow passage on the inner peripheral side thereof, and through a flow passage which is formed to vertically extend from the position 227b to the position 227c of the flow passage in the second layer 250b. In other words, the fluids having passed through the column portions 206, 211 and 217 and the outflow restriction valves 206e, 211e and 217e are introduced to the waste tank 228. A second vent hole 228a communicating with the atmosphere is formed in the upper surface of the first layer 250a at a position corresponding to the waste tank 228.
In the thus-constructed reaction apparatus 90 according to this embodiment, liquids including, e.g., reagents used to develop predetermined reactions, are contained in desired amounts in the liquid container portions of the cartridge 250, and various treatments can be executed by rotationally moving the cartridge 250 in sequence with the motor 37 to selectively change the port-connected position, and by sequentially supplying the liquid from the connected one of the liquid container portions 201-205, 208-210, 213-216 and 218-220 to the reaction vessel 30 with the pump 34 in order to progress a predetermined reaction in the reaction vessel 30, or by transferring the liquids after the reaction to the waster tank 228 from the reaction vessel 30. Particularly, when a reaction product is to be purified, the reaction product is adsorbed on the column while the useless liquid is introduced to the waste tank 228, and a liquid is supplied to the reaction vessel 30 from the liquid container portion 207, 212 or 218 through the column. Further, in the reaction apparatus 90, similarly to the above-described first embodiment illustrated in Fig. 7, the reaction vessel 30 is disposed outside the cartridge 250. Therefore, temperature change of the reaction vessel 30 is less conducted to the cartridge 250, and the reaction vessel 30 and the cartridge 250 can be held at different temperatures (e.g., a reaction temperature and a temperature suitable for preservation, respectively).
The operation of the reaction apparatus 90 according to this embodiment, in particular, the operation for amplifying and labeling genome DNA as a sample will be described below. Fig. 19 is a diagram to explain procedures for amplifying DNA. Fig. 20 is a diagram to explain DNA fragmentation procedures for fragmentizing the amplified DNA and extracting an objective part. Fig. 21 is a diagram to explain procedures for labeling the fragmentized DNA. Those drawings schematically illustrate the liquid container portions and the waste tank 228 of the cartridge 250, the communicating ports connected, and the reaction vessel 30. In those drawings, regarding the liquid container portions 201-205, 207-210, 212-216 and 218 and the waste tank 228, the types and the amounts of liquids contained therein and corresponding numerals illustrated in Fig. 17 are denoted. A blank chamber represents that no liquid is contained therein. Regarding the reaction vessel 30, an oblong circle represents that a liquid is contained in the reaction vessel 30, and a rectangle represents that treatment is executed on the contained liquid. A blank oblong circle represents that no liquid is contained in the reaction vessel 30. Each of arrows in the drawings represents a direction in which a liquid or a gas flows. For the sake of convenience in explanation, step numbers are denoted in association with the reaction vessel 30.
First, a DNA amplifying process is described with reference to Fig. 19. The user puts DNA as a sample to be amplified in the reaction vessel 30, connects the reaction vessel 30 to the rotary disk 82, and fits the rotary disk 82 to the cartridge 250, thus preparing a reaction unit in a similar manner to that in the above-described first embodiment illustrated in Fig. 2B. Then, the user opens the door (not shown) provided in the side of the reaction vessel fixture 36 and places the reaction unit on the rotary stage 38 while laterally sliding the reaction unit into a such state that the top of the reaction vessel 30 is communicated with the delivery/discharge tube 34a and the rotary disk 82 is biased downward by the retainers 84.
At that time, because the retainers 84 are made of Teflon and are flexible, the reaction unit is mounted in a state where a plurality of recesses formed in the bottom surface of the cartridge 250 are engaged with the plurality of projections provided on the upper surface of the rotary stage 38, and where the reaction unit is biased downward by the retainers 84. The user depresses the start button (not shown). Responsively, the CPU 42 in the controller 40 reads and executes a DNA amplifying process routine stored in the flash ROM 43. When that routine is started, the CPU 42 executes control as follows. First, the cartridge 250 is held at a predetermined temperature (e.g., 20°C) by the Peltier device 38a for the cartridge. Then, the motor 37 is driven to rotate the cartridge 250, to thereby communicate the communicating port 201a with the reaction vessel 30, and the pump 34 is operated to lower the air pressure in the reaction vessel 30, to thereby suck the liquid contained in the liquid container portion 201 into the reaction vessel 30 (step S700).
Next, the temperature in the reaction vessel 30 is kept at 4°C by the Peltier device 36a for the reaction vessel, and the pump 34 is operated to continuously lower the air pressure in the reaction vessel 30 and to continuously take the atmosphere into the reaction vessel 30 through the atmosphere flowing portion 221, the liquid container portion 201, the communicating port 201, and the flow path 82a, whereby the mixed liquids contained in the reaction vessel 30 are subjected to bubbling for 10 minutes to develop the reaction of a DNA solution in the reaction vessel 30 (step S710). In such a way, the liquid in the reaction vessel 30 can be subjected to bubbling by continuously taking in the atmosphere through the atmosphere flowing portion 221, any one of the liquid container portions 201-205, 207-210, 212-216 and 218, the communicating port formed at one end of the one of the liquid container portions 201-205, 207-210, 212-216 and 218, and the flow path 82a. The term "bubbling" used in the following description also means such an operation. Further, as illustrated in Fig. 3, the degassing grooves 30a are provided in the reaction vessel 30. When the air pressure in the reaction vessel 30 is lowered and the atmosphere is caused to flow in through the connected port, a rise of a level of the contained liquid and adhesion of the contained liquid to a wall surface are prevented by gases flowing through the degassing grooves 30a, thus ensuring a more satisfactory reaction of the contained liquid. Returning to the explanation of Fig. 19, the communicating port 202a is communicated with the reaction vessel 30 and the liquid contained in the liquid container portion 202 is sucked out (step S720). The temperature in the reaction vessel 30 is kept at 30°C and the bubbling is performed for 30 minutes to develop a reaction of the DNA in the reaction vessel 30 (step S730). Then, the communicating port 203a is communicated with the reaction vessel 30 and the liquid (extraction buffer) contained in the liquid container portion 203 is sucked out (step S740). The temperature in the reaction vessel 30 is kept at 25°C and the bubbling is performed for 5 minutes to develop a reaction (step S750). Then, the communicating port 204a is communicated with the reaction vessel 30 and the liquid (adsorption buffer) contained in the liquid container portion 204 is sucked out for mixing (step S760).
Subsequently, the communicating port 206a is communicated with the reaction vessel 30 and, after changing the connected state of the pump 34, the pump 34 is operated to raise the air pressure in the reaction vessel 30, thus causing the amplified DNA solution in the reaction vessel 30 to flow through the column portion 206 (step S770). Such a situation is described with reference to Fig. 22. Fig. 22 is an explanatory view illustrating a flow route through which the amplified DNA solution flows. When the amplified DNA solution flows through the column portion 206 via the communicating port 206a in the first layer 250a of the cartridge 250 as illustrated in Fig. 22A, only the DNA in a reaction mixture is adsorbed on the column inside the column portion 206. The liquid waste having passed through the column further passes through the outflow restriction valve 206e in the second layer 250b, as illustrated in Fig. 22B, and finally flows into the waste tank 228 after flowing through the vertical flow passage from the position 227c of the flow passage to the first layer 250a. At that time, although the liquid container portion 207 is also connected to the flow route through which the liquid waste flows, the liquid waste is prevented from flowing into the liquid container portion 207 by the inflow restriction valve 206d which is disposed upstream of the liquid container portion 207. Thus, when the product produced by the reaction developed in the reaction vessel 30 is adsorbed on any one of the columns, the product can be adsorbed on the one of the columns and the liquid waste having passed through the relevant column can be caused to flow into the waste tank 228 in a similar manner to that described above except that respective positions of the column portion, the outflow restriction valve, the communicating port, and the vertical flow passage through which the liquid is caused to flow differ depending on which one of the columns is used.
Subsequently, the communicating port 205a is communicated with the reaction vessel 30 and, after changing the connected state of the pump 34, the pump 34 is operated to lower the air pressure in the reaction vessel 30, thereby sucking out the liquid (wash buffer) contained in the liquid container portion 205 (step S780). Then, the communicating port 206a is communicated with the reaction vessel 30 and, after changing the connected state of the pump 34, the pump 34 is operated to raise the air pressure in the reaction vessel 30, thus causing the washing liquid in the reaction vessel 30 to flow through the column portion 16 for washing the column (step S790). The wash buffer after the washing is caused to flow into the waste tank 228 as in step S730. Further, after changing the connected state of the pump 34, the pump 34 is operated to lower the air pressure in the reaction vessel 30 while the communicating port 206a is kept communicated with the reaction vessel 30, whereby the liquid (elution buffer) contained in the liquid container portion 207 is sucked out toward the reaction vessel 30 (step S800) and then introduced to the reaction vessel 30 (step S810) after flowing through the column portion 206. Such a situation is described with reference to Fig. 12. Fig. 12 is an explanatory view illustrating a flow route through which the liquid contained in the liquid container portion 207 flows. When the air pressure in the reaction vessel 30 is lowered, the air pressure in the column portion 206 and the flow route on the side where the liquid waste flows is also lowered. On that occasion, neither the liquid waste nor the gas is sucked out from the waste tank 228 with the provision of the outflow restriction valve 206e. However, the liquid contained in the liquid container portion 207 is sucked out from the liquid container portion 207 and flows through the vertical flow passage from the position 224b of the flow passage at one end of the liquid container portion 207 to the position 224c in the second layer 250b and further through the column portion 206, followed by finally entering the reaction vessel 30. At that time, the amplified DNA adsorbed on the column is eluted into the elution buffer, thus resulting in such a state that a solution including the amplified DNA is contained in the reaction vessel 30. Although the introducing port 207a is formed in the flow passage on the inner peripheral side of the liquid container portion 207, the introducing port 207a is closed by the rotary disk 82 so as to prevent the atmosphere from flowing in and the liquid from flowing out through the introducing port 207a. Thus, when the product adsorbed on any one of the columns is eluted and sucked out from the column, the product is eluted with the liquid contained in the liquid container portion which is connected to the relevant column through the inflow restriction valve, and is then sucked into the reaction vessel 30 in a similar manner to that described above except that respective positions of the column portion, the inflow restriction valve, the communicating port, and the vertical flow passage through which the liquid is caused to flow differ depending on which one of the columns is used. Further, the operation of the pump 34 is continued and the temperature in the reaction vessel 30 is kept at 80°C for 30 minutes to concentrate the amplified DNA solution that has been sucked into the reaction vessel 30 (step S820). The amplified DNA is thereby obtained.
Next, fragmentation of DNA is described with reference to Fig. 20. The CPU 42 in the controller 40 reads and executes a DNA fragmentizing process routine stored in the flash ROM 43. That routine is continuously executed after the end of the DNA amplifying process routine described above. When the DNA fragmentizing process routine is started, the CPU 42 executes control as follows. First, the communicating port 208a is communicated with the reaction vessel 30 to suck out the liquid contained in the liquid container portion 208 (step S900). Then, the temperature in the reaction vessel 30 is kept at 37°C and the bubbling is performed for 1 minute (step S910). Then, the communicating port 209a is communicated with the reaction vessel 30 and the liquid (adsorption buffer) contained in the liquid container portion 209 is sucked out for mixing (step S920). Then, the communicating port 211a is communicated with the reaction vessel 30 and the fragmented DNA solution in the reaction vessel 30 is caused to flow through the column portion 211 (S930). At that time, only the fragmented DNA in a reaction mixture is adsorbed on the column inside the column portion 211, and the liquid waste having passed through the column flows into the waste tank 228. Subsequently, the communicating port 210a is communicated with the reaction vessel 30 and the liquid (wash buffer) contained in the liquid container portion 210 is sucked out (step S940). Then, the communicating port 211a is communicated with the reaction vessel 30 and the liquid in the reaction vessel 30 is caused to flow through the column portion 211 for washing the column (step S950). Further, while keeping the communicating port 211a communicated with the reaction vessel 30, the liquid (elution buffer) contained in the liquid container portion 212 is caused to flow through the column portion 211, following which the elution buffer is sucked toward the reaction vessel 30 (step S960) and then introduced to the reaction vessel 30 (step S970). At that time, the fragmented DNA adsorbed on the column is eluted into the elution buffer, thus resulting in such a state that a solution including the fragmented DNA is contained in the reaction vessel 30. Further, the operation of the pump 34 is continued and the temperature in the reaction vessel 30 is kept at 80°C for 30 minutes to concentrate the fragmented DNA solution that has been sucked into the reaction vessel 30 (step S980). The fragmented DNA is thereby obtained.
Next, labeling of DNA is described with reference to Fig. 21. The CPU 42 in the controller 40 reads and executes a DNA labeling process routine stored in the flash ROM 43. That routine is continuously executed after the end of the DNA fragmentizing process routine described above. When the DNA labeling process routine is started, the CPU 42 executes control as follows. First, the communicating port 213a is communicated with the reaction vessel 30 to suck out the liquid contained in the liquid container portion 213 (step S1000). Then, the temperature in the reaction vessel 30 is kept at 95°C and the bubbling is performed for 5 minutes (step S1010). Then, the temperature in the reaction vessel 30 is kept at 0°C and the bubbling is performed for 3 minutes (step S1020). Then, the communicating port 214a is communicated with the reaction vessel 30 and the liquid contained in the liquid container portion 214 is sucked out (step S1030). Then, the temperature in the reaction vessel 30 is kept at 37°C and the bubbling is performed for 40 minutes (step S1040). Then, the communicating port 215a is communicated with the reaction vessel 30 and the liquid (adsorption buffer) contained in the liquid container portion 215 is sucked out for mixing (step S1050). Then, the communicating port 217a is communicated with the reaction vessel 30 and the fragmented DNA solution in the reaction vessel 30 is caused to flow through the column portion 217 (S1060). At that time, only the labeled DNA fragments in a reaction mixture are adsorbed on the column inside the column portion 217, and the liquid waste having passed through the column flows into the waste tank 228. Subsequently, the communicating port 216a is communicated with the reaction vessel 30 and the liquid (wash buffer) contained in the liquid container portion 216 is sucked out (step S1070). Then, the communicating port 217a is communicated with the reaction vessel 30 and the liquid in the reaction vessel 30 is caused to flow through the column portion 217 for washing the column (step S1080). Further, while keeping the communicating port 217a communicated with the reaction vessel 30, the liquid (elution buffer) contained in the liquid container portion 218 is caused to flow through the column portion 217, following which the elution buffer is sucked toward the reaction vessel 30 (step S1090) and then introduced to the reaction vessel 30. At that time, the labeled DNA adsorbed on the column is eluted into the elution buffer, thus resulting in such a state that a solution including the labeled DNA is contained in the reaction vessel 30. The labeled DNA is thereby obtained.
As described above, the bubbling and the reaction of the liquid(s) contained in the reaction vessel 30 are progressed by connecting any one of the plural liquid container portions 201-205, 207-210, 212-216 and 218 to the reaction vessel 30, applying differential pressure to act upon the liquid in the connected liquid container portion with the operation of the pump 34 to introduce the relevant liquid into the reaction vessel 30, and by continuing the operation of the pump 34 to take the atmosphere, which flows in through the atmosphere flowing passage 221, into the reaction vessel 30. Further, the DNA having been initially put in the reaction vessel 30 can be analyzed by using the labeled DNA obtained through the above-described procedures. The labeled DNA thus obtained is applied to a usual DAN chip, for example. After executing a hybridization reaction step and a washing step, fluorescence emitted from a spot on the DNA chip is captured by a scanner and the intensity of a signal is processed into a numerical value. A change in the number of chromosome copies is measured by setting, as a control, a signal obtained from DNA in a normal cell, and by calculating a ratio of that signal to a signal obtained from DNA in an inspection target cell.
Here, correspondence relations between the components in this embodiment and constituent elements in the present invention are clarified. The cartridge 250 in this embodiment corresponds to a fluid containing cartridge in the present invention, the liquid container portions 201-205, 207-210, 212-216 and 218-220 correspond to container portions, a combination of the first layer 250a and the second layer 250b corresponds to a housing, and the communicating ports 201a-206a, 208a-211a, 213a-217a, 219a and 220a correspond to connecting and communicating portions. Further, each of the first layer 250a and the second layer 250b corresponds to a divided layer, the waste tank 228 corresponds to a reservoir portion, the communicating ports 206a, 211a and 217a correspond to coupling and communicating portions, the outflow restriction valve 206e, 211e and 217e correspond to outflow restricting portions, the inflow restriction valve 206d, 211d and 217d correspond to inflow restriction portions, the water-absorbing porous material corresponds to an absorbing material, the first vent hole 221a corresponds to an atmosphere hole, the hydrophobic porous material corresponds to a porous material, the cartridge mounting mechanism 80 corresponds to mounting unit, the rotation mechanism 32 corresponds to a moving unit, the pump 34 corresponds to the pressure applying unit, the rotary disk 82 corresponds to a contact member, the retainers 84 correspond to a fixing member, the flow path 82a corresponds to a through-hole, the Peltier device 38a for the cartridge corresponds to a cartridge temperature adjusting unit, and the Peltier device 36a for the reaction vessel corresponds to a reaction-vessel temperature adjusting unit.
With the reaction apparatus 90 according to this embodiment, which has been described in detail above, in a state where liquids are contained in the plurality of liquid container portions 201-205, 207-210, 212-216 and 218 which are formed in the first layer 250a and the second layer 250b of the cartridge 250 and which have predetermined volumes determined depending on the liquids to be contained, and where the reaction vessel 30 is connected to any one of the plurality of communicating ports, when pressure is applied to act upon one of the liquid container portions 201-205, 207-210, 212-216 and 218, which is communicated with the communicating port connected to the reaction vessel 30, the atmosphere is supplied to the one of the liquid container portions 201-205, 207-210, 212-216 and 218 through the atmosphere flowing passage 221 communicating with the outside, and the liquid contained in the one liquid container portion is supplied to the reaction vessel 30. Further, the cartridge 250 is rotated to connect another communicating port to the reaction vessel 30 such that plural liquids are eventually supplied to the reaction vessel 30. On that occasion, since the atmosphere supplied through the atmosphere flowing passage 221 is further supplied to the reaction vessel 30 through the communicating port connected to the reaction vessel 30 and the one of the liquid container portions 201-205, 207-210, 212-216 and 218, the plural liquids contained in the reaction vessel 30 are subjected to the bubbling with the inflow of the atmosphere. Accordingly, the contained liquids can be caused to sufficiently develop a reaction in the reaction vessel 30.
Since the ports are disposed in the contact surface, which is formed in the first layer 250a, in the circular pattern coaxial with the central axis of the cartridge 250, it is just required to rotate the cartridge 250 when one of the communicating ports is selectively connected to the reaction vessel 30. Therefore, the connection can be selectively changed with ease, and the liquid can be easily introduced to the reaction vessel 30, thus enabling the reaction to be sufficiently developed with ease. Further, for those ones of the liquid container portions 201-205, 207-210, 212-216 and 218, which are each formed in the shape of a zigzag tube with a zigzag width gradually increasing from the inner peripheral side toward the outer peripheral side of the cartridge 250 having the shape of a circular disk, each port is disposed on the inner peripheral side of the cartridge 250 having the shape of a circular disk. Therefore, the zigzag width can be gradually increased toward the outer peripheral side while effectively utilizing a space in the cartridge 250 having the shape of a circular disk. Also, since the liquid container portions 201-205, 207-210, 212-216 and 218 are each in the tube-like shape, the liquids contained therein can be fully supplied to the reaction vessel 30. For those ones of the liquid container portions 201-205, 207-210, 212-216 and 218, which are each formed in a shape gradually narrowing toward its port, the liquid contained therein can be transferred in amount as possible as close to all, and hence the reaction can be more satisfactorily developed in the reaction vessel 30. For those ones of the chambers, which are each formed in the shape of a tube having narrower opposite ends with liquid flowing portions on the opposite sides formed to be narrower than a liquid containing portion at the middle, the liquid contained in such a chamber can be prevented from flowing out when the cartridge 250 is distributed or handled, for example, and the contained liquid can be caused to sufficiently develop the reaction in the reaction vessel 30 with more ease. For those ones of the chambers, which are each formed in the shape of a longer tube to contain a larger amount of liquid, the liquid can be efficiently contained in such a chamber. Moreover, since the cartridge 250 includes the first layer 250a and the second layer 250b and the chambers are formed in any one of the first layer 250a and the second layer 250b or in a state spreading over both the first layer 250a and the second layer 250b, the chambers can be formed in a larger number. Since the chambers having larger volumes are formed in the second layer 250b, which is positioned away from the contact surface including the ports formed therein, so as to contain larger amounts of liquids than the chambers formed in the first layer 250a, the number of chambers decreases in the layer positioned farther away from the contact surface. Therefore, the number of vertical flow passages connecting the chambers and the ports to each other can be reduced in the second layer 250b, and hence the cartridge 250 is easier to fabricate. Further, since the outflow restriction valves 206e, 211e and 217e, the column portions 206, 211 and 217, and the inflow restriction valves 206d, 211d and 217d are provided, it is possible to, without changing the connection between the reaction vessel 30 and one of the communicating ports 206a, 211a and 217a, adsorb the product produced with the reaction in the reaction vessel 30 on the column inside one of the column portions 206, 211 and 217, to introduce the liquid in one of the liquid container portions 207, 212 and 218 to the corresponding one of the column portions 206, 211 and 217, and then to return the introduced liquid to the reaction vessel 30. Since the water-absorbing porous material capable of absorbing the liquid waste is disposed in the waste tank 228, the liquid waste introduced to the waste tank 228 from the reaction vessel 30 can be more positively held in the waste tank 228 once the liquid waste has been introduced to there. Since the hydrophobic porous material is disposed in the atmosphere flowing portion 221, the liquids contained in those ones of the chambers, which are connected to the atmosphere flowing portion 321, can be prevented from flowing out to the outside through the atmosphere flowing portion 221. Further, for those ones of the chambers, which are connected to the atmosphere flowing portion 221, the liquid in such a chamber can be prevented from flowing out to the outside through the atmosphere flowing portion 221, and the number of first vent holes 21a to be formed can be reduced. In addition, since the reaction vessel 30 is a member having the shape of a tube tapered toward its port, all the liquid can be easily caused to flow between the chamber and the reaction vessel 30 through the port, and the liquid in the reaction vessel 30 can be easily subjected to the bubbling with the atmosphere introduced through the port.
Since the CPU 42 executes the DNA amplifying process routine, the DNA fragmentizing process routine, and the DNA labeling process routine, each routine being set in advance, and controls the rotation mechanism 32 and the pump 34, the liquids can be transferred so as to develop the reaction in accordance with the procedures of the DNA amplifying process routine, the DNA fragmentizing process routine, and the DNA labeling process routine. Comparing with the case where the user performs operations to supply the liquids to the reaction vessel 30 and to develop the reaction at the predetermined temperature for the predetermined time, therefore, each step of the process can be more reliably executed under preset conditions and hence a variation in the reaction results can more positively suppressed. Further, since the liquids are sucked from the liquid container portions 201-205, 207-210, 212-216 and 218 into the reaction vessel 30 by lowering the air pressure in the reaction vessel 30 with the operation of the pump 34 and the liquid waste is pushed out from the reaction vessel 30 to the waste tank 228 by raising the air pressure in the reaction vessel 30, the liquids can be transferred with a comparatively simple arrangement for changing the pressure in the reaction vessel 30. Since the cartridge 250 is rotated about the axis of rotation to selectively connect one of the ports and the reaction vessel 30 to each other, the desired rotation can be more easily performed than the case of rotating the rotary disk 82 to which the delivery/discharge tube 34a is connected. Since the cartridge 250 is mounted in place and the reaction vessel 30 is connected to the rotary disk 82 with the aid of both the rotary disk 82 contacting with the contact surface and the retainers 84 biasing the rotary disk 82 against the contract surface while the cartridge 250 is kept rotatable, the cartridge 250 can be mounted in a state where the chambers and the reaction vessel 30 can be comparatively easily communicated with each other in a selective manner. In addition, with the provision of the Peltier device 38a for the cartridge and the Peltier device 36a for the reaction vessel, it is possible to separately adjust the mounted cartridge 250 to temperature not causing the contained liquids to develop the reaction and the reaction vessel 30 to temperature suitable for the reaction. As a result, the liquids contained in the cartridge 250 can be caused to sufficiently develop the reaction in the reaction vessel 30 regardless of the temperature of the mounted cartridge 250.

[Third Embodiment]

Fig. 24 is an external view of a cartridge 350 according to a third embodiment. Figs. 25 to 28 are plan views illustrating a first layer 351a to a fourth layer 351d of the cartridge 350, respectively. Fig. 29 is a plan view and a front view of a mini-array 350b. Fig. 30 is a partial cross-sectional view, taken along B-B', of the cartridge 350 illustrated in Fig. 24. In Figs. 25 to 28, dotted lines illustrate the structure of a lower surface of the cartridge 350. A reaction apparatus according to this embodiment has a similar structure to that of the reaction apparatus 90 according to the first embodiment except that, unlike the reaction apparatus 90 according to the first embodiment illustrated in Fig. 1, the cartridge 350 is used instead of the cartridge 50, a DNA regulating process routine and a reaction process routine, which are executed by using the cartridge 350 to specify the sort of rice, are stored in the flash ROM 43, the above-described tube pump is used as the pump 34, a pressure gauge (not shown) for detecting pressure in the delivery/discharge tube 34a is attached to the delivery/discharge tube 34a, and that the above-mentioned motor 72 (see Fig. 15) is disposed at a side of the reaction vessel fixture 36 and the above-mentioned rotor 74 (see Fig. 15) is placed in the reaction vessel 30 to be able to perform stirring with the rotor 74 rotated by the motor 72. In the following, therefore, the same components in this third embodiment to those in the reaction apparatus 90 according to the first embodiment are denoted by the same characters and detailed descriptions of those components are omitted. The cartridge 350 includes, though described in detail later, liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 and waste tanks 327 and 328, each capable of containing a liquid, which are collectively referred to as "chambers". This embodiment is described in connection with the case where the reaction apparatus 90 is used to identify the sort of rice from DNA.
The pump 34 used in this embodiment is a tube pump. Therefore, pressure acting upon a target connected to a tube can be raised and lowered by, as required, setting the rotational direction, the number of steps (number of revolutions) and the speed of a stepping motor connected to the pump 34. In the following description of this embodiment, it is assumed that when an operation of introducing a liquid from the reaction vessel 30 to the cartridge 350 and an operation of supplying a liquid from the cartridge 350 to the reaction vessel 30 are switched over, the switching-over is performed by operating the pump 34 after setting the rotational direction, the number of steps (number of revolutions) and the speed of the stepping motor connected to the pump 34. It is also assumed that when the pressure acting upon the target connected to the tube needs to be adjusted, the rotational direction, the number of steps (number of revolutions) and the speed of the stepping motor are set such that an output value of the pressure gauge (not shown) attached to the delivery/discharge tube 34a indicates an objective pressure.
As illustrated in Fig. 24, the cartridge 350 includes a cartridge body 350a for containing liquids necessary to identify the sort of rice, and a mini-array 350b mounted to the cartridge body in a detachable manner.
The cartridge body 350a is a member made of a cycloolefin copolymer and is constituted by four layers, i.e., a first layer 351a to a fourth layer 351d, each of which is formed in the shape of a circular disk. On an upper surface of the first layer 351a, as illustrated in Fig. 25, the cartridge body 350a includes a guide portion 352 to which the rotary disk 82 (see Fig. 2) is fitted. Further, the cartridge body 350a includes, in a lower surface of the fourth layer 351d, three radially extending grooves 342 (having the same role as the grooves 252 in the first embodiment) and a loading hole 341 through which the column is loaded, and further includes guide holes 340c and 340d for attachment of O-rings in the third layer 351c and the fourth layer 351d. Each of the second layer 351b and the third layer 351c of the cartridge body 350a includes a plurality of chambers. As illustrated in Figs. 25 to 28, the cartridge body 350a includes a plurality of liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 capable of containing liquids in predetermined volumes that are determined depending on the liquids to be contained, communicating ports 302a-304a, 308a, 309a, 311a, 315a-321a, 323a and 325a disposed at respective predetermined connecting positions where any one of those liquid container portions is communicated with the reaction vessel 30 when the cartridge 350 is rotationally moved in a selective manner, an atmosphere flowing portion 326 for communicating the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 with the atmosphere to take the atmosphere into the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 or to discharge gases from the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325, waste tanks 327 and 328 capable of containing a liquid waste transferred from the reaction vessel 30, a column portion 306 capable of adsorbing a product produced by a reaction developed in the reaction vessel 30, coupling and communicating ports 306a and 313a disposed at predetermined connecting positions where any of the waste tanks 327 and 328 is connected to the reaction vessel 30 to establish fluid communication therebetween when the cartridge 350 is rotationally moved in a selective manner, closed ports 301a, 305a, 307a, 312a, 322a and 324a each having no hole, a closed flow passage 310 which is not communicated with the atmosphere and which is capable of containing a liquid, and an injection port 310a which is used to inject a liquid to the closed flow passage 310 or to supply the liquid contained in the closed flow passage 310 to the reaction vessel 30.
The liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 are each a space formed in a shape gradually narrowing toward opposite ends. Of the liquid container portions, those ones 304, 308, 309, 315, 316, 318, 319, 321 and 323 containing larger amounts of liquids are each formed as one unitary space spreading over both the second layer 351b and the third layer 351c, and those ones 302, 303, 311, 317, 320 and 325 containing smaller amounts of liquids are formed in only one of the second layer 351b and the third layer 351c. One ends of the liquid container portions 302-304, 308, 309, 311, 315, 316, 318, 319, 321, 323 and 325, which are positioned closer to a center of the cartridge 350, are communicated respectively with communicating ports 302a-304a, 308a, 309a, 311a, 315a, 316a, 318a, 319a, 321a, 323a and 325a through flow passages 302b-304b, 308b, 309b, 311b, 315b, 316b, 318b, 319b, 321b, 323b and 325b, which are formed in a lower surface of the third layer 351c and which are connected to side surfaces of the corresponding liquid container portions near the bottoms thereof, and through vertical flow passages formed in both the third layer 351c and the second layer 351b. One ends of the liquid container portions 317 and 320, which are positioned closer to the center of the cartridge 350, are communicated respectively with communicating ports 317a and 320a through flow passages 317b and 320b, which are formed in a lower surface of the second layer 351b and which are connected to side surfaces of the corresponding liquid container portions near the bottoms thereof, and through vertical flow passages formed in the second layer 351b. The other ends of the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325, which are positioned farther away from the center of the cartridge 350, are each communicated with an atmosphere flowing passage 326. The atmosphere flowing passage 326 will be described in detail later.
The communicating ports 302a-304a, 308a, 309a, 311a, 315a-321a, 323a and 325a are holes which are communicated respectively with the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325, which are used to supply the liquids from the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325, and which are formed in the inner peripheral side of the first layer 351a (i.e., in a contact surface thereof). The communicating ports 302a-304a, 308a, 309a, 311a, 315a-321a, 323a and 325a are disposed on the same plane (flat surface) in a circular pattern coaxial with an axis of rotation about which the cartridge 350 is rotated by the rotation mechanism 32, i.e., with the central axis of the cartridge 350. Further, the liquids contained in the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 can be supplied to the reaction vessel 30 by differential pressure acting upon the liquids contained in the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 which are connected respectively to the associated communicating ports.
The atmosphere flowing portion 326 collectively represents atmosphere flowing passages 302c, 303c, 309c, 311c and 325c which are extended radially outward from respective one ends, positioned farther away from the cartridge center, of the liquid container portions 302, 303, 309, 311 and 325 formed in the lower surface of the third layer 351c, atmosphere flowing passages 317c and 320c which are extended radially outward from respective one ends, positioned farther away from the cartridge center, of the liquid container portions 317 and 320 formed in the lower surface of the second layer 351b, and vent holes 302d-304d, 308d, 309d, 311d, 315d-321d, 323d and 325d which are vertically formed in the first layer 351a. Of the vent holes 302d-304d, 308d, 309d, 311d, 315d-321d, 323d and 325d, those ones 302d, 303d, 309d, 311d and 325d serve to communicate the liquid container portions 302, 303, 309, 311 and 325 with the atmosphere through the atmosphere flowing passages 302c, 303c, 309c, 311c and 325c and through flow passages vertically formed in both the second layer 351b and the third layer 351c. Also, the vent holes 317d and 320d serve to directly communicate the liquid container portions 317 and 320 with the atmosphere through the atmosphere flowing passages 317c and 320c and through flow passages vertically formed in the second layer 351b. Further, the vent holes 304d, 308d, 315d, 316d, 318d, 319d, 321d and 323d serve to communicate the liquid container portions 304, 308, 315, 316, 318, 319, 321 and 323 with the atmosphere without passing through flow passages. A hydrophobic porous material, which allows passing of a liquid, but does not allow passing of the atmosphere, may be disposed in the atmosphere flowing portion 326. The provision of the hydrophobic porous material can prevent the liquids contained in the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 from flowing out to the outside through the atmosphere flowing portion 326. For example, a Teflon porous material (made by Nitto Denko Corporation, TEMISH) can be used as the hydrophobic porous material.
As illustrated in Figs. 26 and 27, the waste tanks 327 and 328 are each a space formed along an outermost periphery of the cartridge 350 as one unitary space spreading over both the second layer 351b and the third layer 351c. The water tank 327 is connected to the column portions 306 through a waste flow passage 327e formed in the second layer 351b to extend radially and connected to the waste tank 327, a flow passage formed to vertically penetrate the second layer 351b from one end of the waste flow passage 327e, which is positioned closer to the center of the cartridge 350, and further through a diffusion flow passage 327f connected to the above vertical flow passage and extending radially. In other words, a fluid having passed through the column portion 306 from the coupling and communicating port 306a is discharged to the waste tank 327. Here, as illustrated in Fig. 27, the diffusion flow passage 327f has substantially the same width as that of the column portion 306. On the other hand, the waste tank 328 is connected to a vertical flow passage 328f, which is formed in the second layer 351b, through a waste flow passage 328e connected to the waste tank 328. Further, vent holes 327d and 328d for communicating the waste tanks 327 and 328 with the atmosphere are formed in the first layer 351a. Inside the waste tanks 327 and 328, a water-absorbing porous material capable of absorbing the liquid waste may be disposed to positively hold the liquid wastes within the waste tanks 327 and 328 once the liquid wastes have flown into there. For example, a sponge or the like can be used as the water-absorbing porous material.
The column portion 306 is disposed between the coupling and communicating port 306a and the diffusion flow passage 327f, and it includes a column. It is here assumed that a ceramic column (e.g., a silica gel) is used as the column. After operating the pump 34 to pressurize the interior of the reaction vessel 30 such that the liquid contained in the reaction vessel 30 flows through the column portion 306 and accumulates in the diffusion flow passage 327f, the liquid accumulated in the diffusion flow passage 327f is introduced to the waste tank 327 by continuing the pressurization. Also, the accumulated liquid is caused to flow through the column portion 306 and to be introduced to the reaction vessel 30 again by depressurizing the interior of the reaction vessel 30. The column can be loaded into the column portion 306 by loading the column from the lower surface side of the fourth layer 351d through the loading hole 341 and then fitting a cover to the lower surface of the fourth layer 451d.
The coupling and communicating ports 306a and 313a are holes which are communicated respectively with the waste tanks 327 and 328, which are used to introduce the liquids to the waste tanks 327 and 328, and which are formed in the inner peripheral side of the first layer 351a (i.e., in the contact surface thereof). The coupling and communicating ports 306a and 313a are disposed on the same plane (flat surface) in a circular pattern coaxial with the axis of rotation about which the cartridge 350 is rotated by the rotation mechanism 32 (see Fig. 1), i.e., with the central axis of the cartridge 350.
The closed ports 301a, 305a, 307a, 312a, 322a and 324a are portions of the first layer 351a where no holes are formed, and their positions are each specified by a packing 354 integrally molded into such a shape that plural O-rings are continuously joined together. When one of those closed ports is positioned to take a state opposed to the reaction vessel 30, the lower surface of the reaction vessel 30 is closed to be able to pressurize or depressurize the liquid that has been sucked into the reaction vessel 30 by the pressurizing or depressurizing operation of the pump 34.
The closed flow passage 310 is formed as a single straight groove in the third layer 351c and is connected to the injection port 310a through a flow passage 310b formed in the third layer 351c to extend radially and through a vertical flow passage formed in both the third layer 351c and the second layer 351b. One end of the closed flow passage 310 on the side away from the center of the cartridge 350 is not connected to the atmosphere flowing portion 326 unlike the above-described liquid container portions. Therefore, when the closed flow passage 310 is not communicated with the reaction vessel 30, the injection port 310a is closed by the lower surface of the rotary disk 82 and hence the closed flow passage 310 becomes an enclosed space.
The injection port 310a is a hole which is communicated with the closed flow passage 310, which is used to introduce the liquid to the closed flow passage 310 or to supply the liquid contained the closed flow passage 310 to the reaction vessel 30, and which is formed in the inner peripheral side of the first layer 351a (i.e., in the contact surface thereof). The injection port 310a is disposed, along with the other ports, on the same plane (flat surface) in the circular pattern coaxial with the axis of rotation about which the cartridge 350 is rotated by the rotation mechanism 32, i.e., with the central axis of the cartridge 350.
As illustrated in Fig. 24, the mini-array 350b is formed to be capable of being removably inserted to a slot 330 which is formed in the third layer 351c and the fourth layer 351d of the cartridge body 350a. Further, a grip 367 is provided in a base end portion of the mini-array 350b such that the mini-array 350b can be easily inserted to or removed from the slot 330.
As illustrated in Figs. 29 and 30, the mini-array 350b includes first and second connecting ports 361 and 362 disposed side by side in a fore end portion thereof, a U-shaped reaction flow passage 365 having two ends connected respectively to the first and second connecting ports 361 and 362, and a spot area 366 disposed in a linear portion of the reaction flow passage 365 at two positions. Further, a slope 368 is provided at the fore end of the mini-array 350b.
In the state where the mini-array 350b is inserted to the slot 330, as illustrated in Fig. 30, the first connecting port 361 is connected to a vertical flow passage 328g, which is formed in the second layer 351b and is communicated with the coupling and communicating port 313a, through a packing 355 in a shape that is obtained by joining two O-rings to each other. Also, the second connecting port 362 is connected to the vertical flow passage 328f (see also Fig. 26), which is formed in the second layer 351b, through the packing 355. Fig. 30A illustrates a cross-section of the slot 330 before the mini-array 350b is inserted to the slot 330. When the mini-array 350b is inserted to the slot 330, the packing 355 does not interfere with the insertion of the mini-array 350b because the slope 368 formed in the mini-array 350b first contacts with the packing 355. The packing 355 is fitted from the lower surface side of the fourth layer 351d through the guide holes 340c (see also Fig. 27) and 340d (see also Fig. 28) which are formed respectively in the third layer 351c and the fourth layer 351d.
As illustrated in Fig. 29, the spot area 366 of the mini-array 350b includes a plurality of DNA spots formed therein to cause hybridization when a DNA solution flows into there from the reaction vessel 30 through the first connecting port 361. More specifically, in the spot area 366, the DNA spots are arrayed in plural rows at constant intervals not only in the widthwise direction of the reaction flow passage 365, but also in the lengthwise direction thereof. Further, as illustrated in Figs. 29 and 30, the mini-array 350b is formed such that the reaction flow passage 365 is located at a lower position when the mini-array 350b is inserted to the slot 330. Therefore, the temperature of the liquid in the reaction flow passage 365 can be easily adjusted by the Peltier device 38a for the cartridge, which is installed in the rotary stage 38.
In the thus-constructed reaction apparatus 90 according to this embodiment, the cartridge 350 is used in a state that the mini-array 350b is previously inserted to the cartridge by 350a. In the cartridge 350, liquids including, e.g., reagents used to develop predetermined reactions, are contained in desired amounts in the liquid container portions of the cartridge 350. The cartridge 350 is rotationally moved in sequence by the motor 37 to selectively change the port position of the cartridge 350 as appropriate, which is to be connected to the reaction vessel 30, for sequentially supplying the liquid from the connected one of the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 to the reaction vessel 30 in order to progress a predetermined reaction in the reaction vessel 30, or for transferring the liquids after the reaction to the waster tanks 327 and 328 from the reaction vessel 30. Particularly, when a reaction product is to be purified, the reaction product is adsorbed on the column while the useless liquid is discharged to the waste tank 327. Further, the reaction product adsorbed on the column is eluted with the liquid contained in any one of the liquid container portions, the liquid including the eluted product is temporarily accumulated in the diffusion flow passage 327f, and the eluted product is supplied to the reaction vessel 30 again. Moreover, in the reaction apparatus 90, similarly to the above-described first embodiment illustrated in Fig. 7, the reaction vessel 30 is disposed outside the cartridge 350. Therefore, temperature change of the reaction vessel 30 is less conducted to the cartridge 350, and the reaction vessel 30 and the cartridge 350 can be held at different temperatures (e.g., a reaction temperature and a temperature suitable for preservation, respectively).
The operation of the reaction apparatus 90 according to this embodiment, in particular, the operation for amplifying and regulating genome DNA of rice as a sample and reacting the regulated genome DNA with the DNA spots formed on the spot area 366 of the mini-array 350b will be described below. Fig. 31 is a diagram to explain procedures for amplifying and regulating genome DNA of rice, and Fig. 32 is a diagram to explain procedures for reacting the regulated genome DNA with the DNA spots, which are formed in the spot area 366 of the mini-array 350b. Those drawings schematically illustrate the liquid container portions and the waste tanks 327 and 328 of the cartridge 350, the communicating ports connected, the injection port, and the reaction vessel 30. In those drawings, regarding the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 and the waste tanks 327 and 328, the types and the amounts of liquids contained therein and corresponding numerals illustrated in Figs. 25 to 28 are denoted. A blank chamber represents that no liquid is contained therein. Regarding the reaction vessel 30, an oblong circle represents that a liquid is contained in the reaction vessel 30, and a rectangle represents that treatment is executed on the contained liquid. A blank oblong circle represents that no liquid is contained in the reaction vessel 30. Each of arrows in the drawings represents a direction in which a liquid or a gas flows. For the sake of convenience in explanation, step numbers are denoted in association with the reaction vessel 30.
First, a DNA amplifying and regulating process is described with reference to Figs. 1, 2 and 31. First, the user puts genome DNA of rice, of which sort is to be identified, in the reaction vessel 30, connects the reaction vessel 30 to the rotary disk 82, and fits the rotary disk 82 to the cartridge 350, thus preparing a reaction unit in a similar manner to that in the above-described first embodiment illustrated in Fig. 2B. Then, the user opens the door (not shown) provided in the side of the reaction vessel fixture 36 and places the reaction unit on the rotary stage 38 while laterally sliding the reaction unit into a such state that the top of the reaction vessel 30 is communicated with the delivery/discharge tube 34a and the rotary disk 82 is biased downward by the retainers 84. At that time, because the retainers 84 are made of Teflon and are flexible, the reaction unit is mounted in a state where a plurality of recesses formed in the bottom surface of the cartridge 350 are engaged with the plurality of projections provided on the upper surface of the rotary stage 38, and where the reaction unit is biased downward by the retainers 84. The user depresses the start button (not shown). Responsively, the CPU 42 in the controller 40 reads and executes the DNA regulating process routine stored in the flash ROM 43. When that routine is started, the CPU 42 executes control as follows. First, the motor 37 is driven to rotate the cartridge 350, to thereby communicate the communicating port 302a with the reaction vessel 30, and the pump 34 is operated to lower the air pressure in the reaction vessel 30, to thereby suck the liquid contained in the liquid container portion 302 into the reaction vessel 30 (step S1100).
Next, the communicating port 303a is communicated with the reaction vessel 30 and the pump 34 is operated to suck out the liquid contained in the liquid container portion 303 (step S1110). After rotating the cartridge 350 so as to connect the closed port 305a to the reaction vessel 30, the temperature in the reaction vessel 30 is kept at 95°C and a reaction is developed under stirring for 15 minutes. The reaction is then continued by repeating 40 cycles of stirring for 1 minute with the temperature in the reaction vessel 30 kept at 95°C, stirring for 1 minute and 30 seconds at the temperature of 66°C, and stirring for 30 seconds at the temperature of 72°C, and by finally performing stirring for 10 minutes at the temperature of 72°C (step S1120). Here, the term "stirring" means an operation of mixing a solution in the reaction vessel 30 by rotating the rotor 47, which is put in the reaction vessel 30, with the motor 47. Then, the communicating port 304a is communicated with the reaction vessel 30 and the pump 34 is operated to suck out the liquid (adsorption buffer (3.8 mol/L ammonium sulfate)) contained in the liquid container portion 304 (step S1130). Then, the coupling and communicating port 306a is communicated with the reaction vessel 30 and the pump 34 is operated to cause the mixed solution in the reaction vessel 30 to flow through the column portion 306 (step S1140). When the mixed solution flows through the column portion 306 via the coupling and communicating ports 306a in the first layer 351a of the cartridge 350, only the DNA in a reaction mixture is adsorbed on the column inside the column portion 306. The liquid waste having passed through the column finally flows into the waste tank 327, as described above, via the diffusion flow passage 327f illustrated in Fig. 27.
Subsequently, the communicating port 323a is communicated with the reaction vessel 30 and the pump 34 is operated to suck out the liquid (first wash buffer (1.9 mol/L ammonium sulfate)) contained in the liquid container portion 323. The temperature in the reaction vessel 30 is kept at 25°C and the interior of the reaction vessel 30 is washed under the stirring for 1 minute (step S1150). The reason why the interior of the reaction vessel 30 is washed here is to prevent precipitation of salt. Then, the pump 34 is operated to introduce the liquid in the reaction vessel 30 after the washing to the liquid container portion 323. Then, the communicating port 308a is communicated with the reaction vessel 30 and the pump 34 is operated to suck out the liquid (second wash buffer (pH 6.0, 10 mmol/L phosphoric acid - ethanol mixed solution (mixing ratio of 1 : 2.8)) (step S1170). Then, the coupling and communicating port 306a is communicated with the reaction vessel 30 and the pump 34 is operated to cause the second wash buffer in the reaction vessel 30 to flow through the column portion 306 for washing the column (step S1180). Then, the communicating port 309a is communicated with the reaction vessel 30 and the pump 34 is operated to suck out the liquid (elution buffer (pH 8.0, 20 mmol/L tris-hydrochloric acid)) contained in the liquid container portion 309 (step S1190). Then, the coupling and communicating port 306a is communicated with the reaction vessel 30 and the pump 34 is operated to cause the elution buffer in the reaction vessel 30 to flow through the column portion 306, and the eluate is kept accumulated in the diffusion flow passage 327f without flowing out to the waste tank 327 (step S1200). In practice, after causing the elution buffer to flow through the column portion 306, the operation of squeezing a tube by the pump 34 (tube pump) is stopped. On that occasion, the amplified DNA adsorbed on the column is eluted into the elution buffer, thus resulting in such a state that a solution containing the amplified DNA is accumulated in the diffusion flow passage 327f.
The diffusion flow passage 327f has substantially the same width as that of the column portion 306 as described above, and the degree of diffusion of the DNA eluted in the elution buffer after flowing through the column is greater than the case of using a flow passage of which width is smaller than that of the diffusion flow passage 327f. Therefore, distribution of the DNA is less localized in the elution buffer. The reason why the width of the diffusion flow passage 327f is set substantially the same as that of the column portion 306 is as follows. When the elution buffer is caused to flow through the column portion 306, the adsorbed DNA is eluted in a larger amount into part of the elution buffer which has flown through the column portion 306 earlier, while the adsorbed DNA is less eluted into part of the elution buffer which has flown through the column portion 306 later, because a large amount of the adsorbed DNA is already eluted. Accordingly, if the width of the diffusion flow passage 327f is set smaller than that of the column portion 306, the DNA concentration becomes lower in a portion of the diffusion flow passage 327f closer to the column portion 306 and higher in a portion thereof farther away from the column portion 306 after the end of the elution with the elution buffer. When the DNA is sucked back into the reaction vessel 30 together with the elution buffer, the elution buffer is caused to flow through the column portion 306 in a direction reversal to that when the DNA has been eluted. Namely, the DNA concentration becomes higher in part of the elution buffer, which flows back through the column portion 306 later. Hence, the DNA is apt to remain in the column portion 306 at the time after returning the elution buffer into the reaction vessel 30. In contrast, by setting the width of the diffusion flow passage 327f to be substantially the same as that of the column portion 306, the DNA concentration is diffused and is less localized even if the DNA concentration is higher in part of the elution buffer which has flown through the column portion 306 earlier and is lower in part of the elution buffer which has flown through the column portion 306 later. As a result, a recovery rate of DNA can be increased.
After step S1200, the pump 34 is operated to suck the elution buffer, which is accumulated in the diffusion flow passage 327f and which includes the eluted DNA, for return to the reaction vessel 30 (step S1210). At that time, since the width of the diffusion flow passage 327f is substantially the same as that of the column portion 306, the eluted DNA is diffused to a larger extent than the case where the width of the diffusion flow passage 327f is smaller than that of the column portion 306, and the eluted DNA is less apt to remain in the column portion 306 when the elution buffer is sucked into the reaction vessel 30 together with the eluted DNA. Then, the injection port 310a is communicated with the reaction vessel 30 and the pump 34 is operated to inject the elution buffer in the reaction vessel 30 to the closed flow passage 310 (step S1220). On that occasion, air filled in the closed flow passage 310 compressed by the injected liquid into a higher-pressure state. Here, by adjusting the pressure applied to the reaction vessel 30 with the pump 34, the amount of the mixed solution injected into the closed flow passage 310 can be adjusted depending on the volume of the closed flow passage 310 and the applied pressure. Assuming the pressure to be 202 kPa (2 atm), for example, the mixed solution can be injected in amount corresponding to half the volume of the closed flow passage 310. Then, the communicating port 309a is communicated with the reaction vessel 30 and the mixed solution remaining in the reaction vessel 30 is discharged to the liquid container portion 309 (step S1230). At that time, because the pressure having been applied to inject the mixed solution to the closed flow passage 310 in step S1220 still remains in the reaction vessel 30, the mixed solution in the reaction vessel 30 is discharged to the liquid container portion 309 by the action of the remaining pressure when the communicating port 309a is communicated with the reaction vessel 30. Then, the injection port 310a is communicated with the reaction vessel 30 and the mixed solution having been injected to the reaction flow passage 310 is supplied to the reaction vessel 30 (step S1240), thereby obtaining the regulated DNA. On that occasion, because the mixed solution has been discharged to the liquid container portion 309 in step S1240 by the action of the pressure remaining in the reaction vessel 30, the pressure in the reaction vessel 30 is lowered while the pressure of air in the closed flow passage 310 is still kept at a level when the mixed solution has been injected to there in step S1220. Therefore, the mixed solution having been injected to the closed flow passage 310 is supplied to the reaction vessel 30 under such a pressure difference. The mixed solution may be supplied to the reaction vessel 30 by operating the pump 34 such that the mixed solution in the liquid container portion 309 is reliably supplied to the reaction vessel 30.
Next, the procedures for reacting the regulated DNA with the DNA spots, which are formed in the spot area 366 of the mini-array 350b, will be described with reference to Fig. 32.
The CPU 42 in the controller 40 reads and executes a reaction process routine stored in the flash ROM 43. That routine is continuously executed after the end of the DNA regulating process routine described above. When the reaction process routine is started, the CPU 42 executes control as follows. First, the communicating port 311a is communicated with the reaction vessel 30 including the regulated DNA, and the pump 34 is operated to suck out the liquid contained in the liquid container portion 311 (step S1300). After rotating the cartridge 350 so as to connect the closed port 312a to the reaction vessel 30, the temperature in the reaction vessel 30 is kept at 90°C and the stirring is performed for 5 minutes (step S1310). Then, the temperature in the reaction vessel 30 is kept at 10°C and the stirring is performed for 5 minutes (step S1320). Then, the coupling and communicating port 313a is communicated with the reaction vessel 30 and the operation of the pump 34 is adjusted such that the mixed solution contained in the reaction vessel 30 is temporarily accumulated in the reaction flow passage 365 of the mini-array 350b. The temperature in the reaction flow passage 365 is held at 42°C for 60 minutes by the Peltier device 38a for the cartridge to develop a hybridization reaction between probe DNAs in the DNA spots placed in the spot area 366 and the DNA in the mixed solution, and the pump 34 is then operated again to raise the air pressure in the reaction vessel 30 such that the liquid temporarily accumulated in the reaction flow passage 365 is discharged to the waste tank 328 (step S1330). On that occasion, the mixed solution having flown through the mini-array 350b is introduced to the waste tank 328 through the above-described route.
Subsequently, the communicating port 315a is communicated with the reaction vessel 30 and the pump 34 is operated to suck out the liquid contained in the liquid container portion 315 (step S1340). Then, the coupling and communicating port 313a is communicated with the reaction vessel 30 and the operation of the pump 34 is adjusted such that the washing liquid contained in the reaction vessel 30 is temporarily accumulated in the reaction flow passage 365 of the mini-array 350b. The temperature in the reaction flow passage 365 is held at 25°C for 5 minutes by the Peltier device 38a for the cartridge to wash the spot area 366, and the pump 34 is then operated again to raise the air pressure in the reaction vessel 30 such that the washing liquid temporarily accumulated in the reaction flow passage 365 is discharged to the waste tank 328 (step S1350). Thereafter, similar processes to those in steps S1340 and step S1350 are executed by using the liquid contained in the liquid container portion 316 to wash the spot area 366 of the mini-array 350b (steps S1360 to S1370). Then, the communicating port 317a is communicated with the reaction vessel 30 and the pump 34 is operated to suck out the liquid contained in the liquid container portion 317 (step S1380). Then, the coupling and communicating port 313a is communicated with the reaction vessel 30 and the operation of the pump 34 is adjusted such that the liquid contained in the reaction vessel 30 is temporarily accumulated in the reaction flow passage 365 of the mini-array 350b. The temperature in the reaction flow passage 365 is held at 25°C for 30 minutes to develop a chemical luminescent reaction of the DNA in the spot area 366, and the pump 34 is then operated again to raise the air pressure in the reaction vessel 30 such that the liquid temporarily accumulated in the reaction flow passage 365 is discharged to the waste tank 328 (step S1390). Thereafter, similar processes to those in steps S1340 and step S1350 are executed by using the liquids contained in the liquid container portions 318 and 319 to wash the spot area 366 of the mini-array 350b (steps S1400 to S1430). Then, the communicating port 320a is communicated with the reaction vessel 30 and the pump 34 is operated to suck out the liquid contained in the liquid container portion 320 (step S1440). Then, the coupling and communicating port 313a is communicated with the reaction vessel 30 and the operation of the pump 34 is adjusted such that the liquid contained in the reaction vessel 30 is temporarily accumulated in the reaction flow passage 365 of the mini-array 350b. The temperature in the reaction flow passage 365 is held at 25°C for 30 minutes to develop a pigment deposition reaction of the DNA in the spot area 366, and the pump 34 is then operated again to raise the air pressure in the reaction vessel 30 such that the liquid temporarily accumulated in the reaction flow passage 365 is discharged to the waste tank 328 (step S1450). Then, the communicating port 321a is communicated with the reaction vessel 30 and the pump 34 is operated to suck out the liquid contained in the liquid container portion 321 (step S1460). Then, the coupling and communicating port 313a is communicated with the reaction vessel 30 and the liquid contained in the reaction vessel 30 is caused to flow through the reaction flow passage 365 of the mini-array 350b, thereby stopping the pigment deposition reaction of the DNA in the spot area 366 (step S1470). As a result, the DNA spots where pigments have deposited are obtained in the mini-array 350b (step S1480). An image of the mini-array 350b is scanned by using an OA scanner (GT-8700F, made by Epson Company), and the sort of rice is determined from a pigment deposition pattern that has appeared on the scanned image. The pigment deposition pattern can also be visually determined.
Here, correspondence relations between the components in this embodiment and constituent elements in the present invention are clarified. The cartridge 350 in this embodiment corresponds to a fluid containing cartridge in the present invention, the communicating ports 302a-304a, 308a, 309a, 311a, 315a-321a, 323a and 325a correspond to connecting and communicating portions, the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 correspond to container portions, and a combination of the first layer 351a to the fourth layer 351d corresponds to a housing. Further, each of the first layer 351a to the fourth layer 351d corresponds to a divided layer, each of the waste tanks 327 and 328 corresponds to a reservoir portion, the coupling and communicating ports 306a and 313a correspond to coupling and communicating portions, the vent holes 302d-304d, 308d, 309d, 311d, 315d-321d, 323d and 325d correspond to atmosphere holes, the cartridge mounting mechanism 80 corresponds to a mounting unit, the rotation mechanism 32 corresponds to a moving unit, the pump 34 corresponds to the pressure applying unit, the rotary disk 82 corresponds to a contact member, the retainers 84 correspond to a fixing member, the flow path 82a corresponds to a through-hole, the Peltier device 38a for the cartridge corresponds to a cartridge temperature adjusting unit, and the Peltier device 36a for the reaction vessel corresponds to a reaction-vessel temperature adjusting unit.
With the reaction apparatus 90 according to this embodiment, which has been described in detail above, when one of the communicating ports, which are disposed in the circular pattern coaxial with the central axis of the cartridge 350, is selectively connected to the reaction vessel 30, it is just required to rotate the cartridge 350. Therefore, the connection can be selectively changed with ease, and the liquid can be easily introduced to the reaction vessel 30, thus enabling the reaction to be sufficiently developed with ease. Further, for those ones of the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325, which are each formed in a shape gradually narrowing toward its port, the liquid contained therein can be transferred in amount as possible as close to all, and hence the reaction can be more satisfactorily developed in the reaction vessel 30. For those ones of the chambers, which are each formed in the shape of a longer tube to contain a larger amount of liquid, the liquid can be efficiently contained in such a chamber. Moreover, since the cartridge 350 includes the first layer 351a to the fourth layer 351d and the chambers are formed in any one of the second layer 351b and the third layer 351c or in a state spreading over both the second layer 351b and the third layer 351c, the chambers can be formed in a larger number.
Since the CPU 42 executes the DNA regulating process routine and the DNA reaction process routine, each routine being set in advance, and controls the rotation mechanism 32 and the pump 34, the liquids can be transferred so as to develop the reaction in accordance with the procedures of the DNA regulating process routine and the DNA reaction process routine. Comparing with the case where the user performs operations to supply the liquids to the reaction vessel 30 and to develop the reaction at the predetermined temperature for the predetermined time, therefore, each step of the process can be more reliably executed under preset conditions and hence a variation in the reaction results can more positively suppressed. Further, since the liquids are sucked from the liquid container portions 302-304, 308, 309, 311, 315-321, 323 and 325 into the reaction vessel 30 by lowering the air pressure in the reaction vessel 30 with the operation of the pump 34 and the liquid waste is pushed out from the reaction vessel 30 to the waste tank 228 by raising the air pressure in the reaction vessel 30, the liquids can be transferred with a comparatively simple arrangement for changing the pressure in the reaction vessel 30. Since the cartridge 350 is rotated about the axis of rotation to selectively connect one of the ports and the reaction vessel 30 to each other, the desired rotation can be more easily performed than the case of rotating the rotary disk 82 to which the delivery/discharge tube 34a is connected. Since the cartridge 350 is mounted in place and the reaction vessel 30 is connected to the rotary disk 82 with the aid of both the rotary disk 82 contacting with the contact surface and the retainers 84 biasing the rotary disk 82 against the contract surface while the cartridge 350 is kept rotatable, the cartridge 350 can be mounted in a state where the chambers and the reaction vessel 30 can be comparatively easily communicated with each other in a selective manner. In addition, with the provision of the Peltier device 38a for the cartridge and the Peltier device 36a for the reaction vessel, it is possible to separately adjust the mounted cartridge 350 to temperature not causing the contained liquids to develop the reaction and the reaction vessel 30 to temperature suitable for the reaction. As a result, the liquids contained in the cartridge 350 can be caused to sufficiently develop the reaction in the reaction vessel 30 regardless of the temperature of the mounted cartridge 350.
Be it noted that the present invention is in no way limited to the above-described third embodiment and can be carried out in various embodiments within the technical scope of the present invention.
For example, while in the above-described third embodiment the cartridge 350 and the reaction apparatus 90 are used to identify the sort of rice, the present invention may also be applied to other chemical reactions. For example, the cartridge may contain liquids necessary for executing post-treatment of a CGH array, and the reaction apparatus may execute a chemical reaction in accordance with a routine for the post-treatment of the CGH array, i.e., a CGH post-treatment routine. Fig. 33 is an external view of a cartridge 450 for the post-treatment of the CGH array. Figs. 34 to 36 are respective plan views of a first layer 451a to a third layer 451c of a cartridge body 450a. Fig. 37 is a diagram to explain procedures for reacting labeled DNA with DNA spots formed in a spot area of a mini-array 450b for the CGH post-treatment. In Figs. 34 to 36, dotted lines illustrate the structure of a lower surface of the cartridge 450.
As illustrated in Fig. 33, the cartridge 450 includes a cartridge body 450a for containing liquids that are used in the post-treatment of the CGH array, and a mini-array 450b mounted to the cartridge body in a detachable manner.
The cartridge body 450a is a member made of a cycloolefin copolymer and is constituted by three layers, i.e., a first layer 451a to a third layer 451c, each of which is formed in the shape of a circular disk. On an upper surface of the first layer 451a, as illustrated in Fig. 34, the cartridge body 450a includes a guide portion 452 to which the rotary disk 82 (see Fig. 2) is fitted. Further, the cartridge body 450a includes three radially extending grooves 442 (having the same role as the grooves 252 in the first embodiment) in a lower surface of the fourth layer 451d and a guide hole 440c for attachment of an O-ring in the third layer 451c. As illustrated in Figs. 34 to 36, the cartridge body 450a includes a plurality of liquid container portions 412, 415, 417, 419, 421 and 423, communicating ports 412a, 415a, 417a, 419a, 421a and 423a, vent holes 412d, 415d, 417d, 419d, 421d, 423d and 428d, a waste tank 428, a coupling and communicating port 413a, and closed ports 401a-411a, 416a, 418a, 312a, 420a, 422a, 424a and 425a. One ends of the liquid container portions 412, 415, 417, 419, 421 and 423, which are positioned closer to the center of the cartridge body 450a, are communicated respectively with communicating ports 412a, 415a, 417a, 419a, 421a and 423a, and the other ends of those liquid container portions, which are positioned farther away from the center of the cartridge body 450a, are communicated respectively with the vent holes 412d, 415d, 417d, 419d, 421d and 423d. One end of the waste tank 428 is communicated with the coupling and communicating port 413a through a waste flow passage 428a and the mini-array 450b inserted to a slot 430, and the other end of the waste tank 428 is directly communicated with a vent hole 428d. The closed ports 401a-411a, 416a, 418a, 312a, 420a, 422a, 424a and 425a are portions of the first layer 451a where no holes are formed, and their positions are each specified by a packing 454 integrally molded into such a shape that plural O-rings are continuously joined together.
The CGH array post-treatment using the cartridge body 450a will be described with reference to Figs. 1, 2 and 37. First, the user puts the labeled DNA as a sample in the reaction vessel 30 and places it on the rotary stage 38 in a similar manner to that in the above-described first embodiment. Then, the user depresses the start button (not shown). Responsively, the CPU 42 in the controller 40 reads and executes a CGH array post-treatment routine stored in the flash ROM 43. When that routine is started, the CPU 42 executes control as follows. First, the communicating port 412a is communicated with the reaction vessel 30, and the pump 34 is operated to suck out the liquid contained in the liquid container portion 412 (step S1500). After rotating the cartridge 450 so as to connect the closed port 411a to the reaction vessel 30, the temperature in the reaction vessel 30 is kept at 90°C and the stirring is performed for 5 minutes (step S1510). Then, the temperature in the reaction vessel 30 is kept at 10°C and the stirring is performed for 5 minutes (step S1520). Then, the coupling and communicating port 413a is communicated with the reaction vessel 30 and the operation of the pump 34 is adjusted such that the mixed solution contained in the reaction vessel 30 is temporarily accumulated in a reaction flow passage of the mini-array 450b. The temperature in the reaction flow passage is held at 42°C for 240 minutes by the Peltier device 38a for the cartridge to develop a hybridization reaction between probe DNAs in DNA spots disposed in a spot area of the mini-array 450b and the DNA in the mixed solution, and the pump 34 is then operated again to raise the air pressure in the reaction vessel 30 such that the liquid temporarily accumulated in the reaction flow passage is introduced to the waste tank 428 (step S1530). At that time, the mixed solution having flown through the mini-array 450b is introduced to the waste tank 428 through the above-described route.
Subsequently, the communicating port 415a is communicated with the reaction vessel 30 and the pump 34 is operated to suck out the liquid contained in the liquid container portion 415 (step S1540). Then, the coupling and communicating port 413a is communicated with the reaction vessel 30 and the operation of the pump 34 is adjusted such that the washing liquid contained in the reaction vessel 30 is temporarily accumulated in the reaction flow passage of the mini-array 450b. The temperature in the reaction flow passage is held at 42°C for 1 minute by the Peltier device 38a for the cartridge to wash the spot area 366 of the mini-array 450b, and the pump 34 is then operated again to raise the air pressure in the reaction vessel 30 such that the washing liquid temporarily accumulated in the reaction flow passage 365 is discharged to the waste tank 428 (step S1550). Thereafter, similar processes to those in steps S1540 and step S1550 are executed by using the liquid contained in the liquid container portion 417 to wash the spot area of the mini-array 450b (steps S1560 to S1570). Then, similar processes to those in steps S1540 and step S1550 except that the time of holding the temperature in the reaction flow passage of the mini-array 450b at 42°C by the Peltier device 38a for the cartridge is 2 minutes are executed by using the liquid contained in the liquid container portion 419 to wash the spot area of the mini-array 450b (steps S1580 to S1590). Then, similar processes to those in steps S1540 and step S1550 are executed by using the liquids contained in the liquid container portions 421 and 423 to wash the spot area of the mini-array 450b (steps S1600 to S1630). As a result, the DNA spots after the hybridization reaction are obtained in the mini-array 450b (step S1640). The mini-array 450b having the thus-obtained DNA spots after the hybridization reaction is set in an adapter for a dedicated scanner, for example, such that fluorescence emitted from each of the DNA spots is captured by the scanner and the intensity of a signal is processed into a numerical value.
In the above-described third embodiment, the regulated DNA is obtained (steps S1220-S1240) by, after the process of step S1210 in Fig. 31, supplying the mixed solution in the adjusted amount to the reaction vessel 30 by employing the closed flow passage 310. However, the regulated DNA may be obtained by, after the process of step S1210, concentrating the mixed solution in the reaction vessel 30 through the steps of rotating the cartridge 350 so as to connect the closed port 312a to the reaction vessel 30, and operating the pump 34 to increase the air pressure in the reaction vessel 30 to 70 kPa, while the temperature in the reaction vessel 30 is held at 80°C for 50 minutes, on condition that the rotational direction, the number of steps (number of revolutions) and the speed of the stepping motor connected to the pump 34 are proper set.
This application claims a priority based on Japanese Patent Application No. 2007-323915 filed on December 14, 2007 , Japanese Patent Application No. 2008-061051 filed on March 11, 2008 , Japanese Patent Application No. 2008-172659 filed on July 1, 2008 , and Japanese Patent Application No. 2007-323914 filed on December 14, 2007 , the entire contents of which are incorporated herein by reference.

Industrial Applicability

The present invention can be applied to not only the analysis of genes, but also various chemical reactions.

Claims

A fluid containing cartridge for containing fluids in an extractable manner, the fluid containing cartridge comprising:
a plurality of container portions being able to contain the fluids and formed inside a housing in respective predetermined volumes that are determined depending on the fluids to be contained therein;

an atmosphere flowing portion for communicating the container portions with the outside, thus enabling the atmosphere to flow into the container portions; and

a plurality of connecting and communicating portions disposed at respective predetermined connecting positions where, by relatively moving a reaction vessel disposed in the outside and capable of containing the fluids and the fluid containing cartridge in a selective manner, the reaction vessel and any one of the container portions are connected to each other for fluid communication therebetween, the connecting and communicating portions enabling the fluids contained in the container portions to be supplied to the reaction vessel by differential pressure acting upon the fluids contained in the container portions.
The fluid containing cartridge according to Claim 1, wherein the plurality of connecting and communicating portions are disposed on a flat surface formed in the housing in a circular pattern coaxial with an axis of rotation about which any one of the reaction vessel and the housing is rotated.
The fluid containing cartridge according to Claim 2, wherein at least one of the plurality of container portions is formed in the housing having the shape of a circular disk and has the shape of a zigzag tube with a zigzag width gradually increasing from an inner peripheral side toward an outer peripheral side of the circular disk-shaped housing,
wherein the plurality of connecting and communicating portions are disposed in the inner peripheral side of the circular disk-shaped housing.
The fluid containing cartridge according to Claim 2 or 3, wherein the plurality of container portions are formed in the housing having the shape of a circular disk such that the container portions containing the fluids in larger amounts are positioned in an even outer peripheral side of the circular disk-shaped housing.
The fluid containing cartridge according to any one of Claims 1 to 4, wherein at least one of the plurality of container portions is formed in the shape of a tube gradually narrowing toward the connecting and communicating portion.
The fluid containing cartridge according to any one of Claims 1 to 5, wherein at least one of the plurality of container portions is formed in the shape of a tube gradually narrowing toward opposite ends thereof.
The fluid containing cartridge according to any one of Claims 1 to 6, wherein the plurality of container portions are formed such that the container portions containing the fluids in larger amounts have the shapes of longer tubes.
The fluid containing cartridge according to any one of Claims 1 to 7, wherein the housing comprises a plurality of divided layers, and
wherein the container portions are formed in any one of the divided layers, or formed in a state extending over two or more of the divided layers.
The fluid containing cartridge according to Claim 8, wherein the plurality of container portions are formed such that the container portions formed in the divided layer positioned farther away from the flat surface, in which the connecting and communicating portions are formed, are able to contain the fluids in larger amounts.
The fluid containing cartridge according to any one of Claims 1 to 9, further comprising:
a reservoir portion being able to reserve a fluid and formed in the housing to be communicated with the atmosphere; and

a coupling and communicating portion disposed at a predetermined coupling position where the reaction vessel and the reservoir portion are communicated with each other when the reaction vessel and the fluid containing cartridge are relatively moved into a state of predetermined positional relation in a selective manner.
The fluid containing cartridge according to Claim 10, further comprising:
an outflow restriction portion disposed between the reservoir portion and the coupling and communicating portion to allow flow of the fluid from the reaction vessel to the reservoir portion and to block off flow of the fluid from the reservoir portion to the reaction vessel;

a column portion disposed between the coupling and communicating portion and the outflow restriction portion and being able to adsorb a product produced in the reaction vessel; and

an inflow restriction portion disposed between any one of the plurality of container portions and the column portion to allow flow of the fluid from the one container portion to the column portion and to block off flow of the fluid from the column portion to the one container portion.
The fluid containing cartridge according to Claim 11, wherein the column portion includes a ceramic column.
The fluid containing cartridge according to Claim 11 or 12, wherein an absorbing material for absorbing the fluid is disposed in the reservoir portion.
The fluid containing cartridge according to any one of Claims 1 to 13, wherein the container portions contain, as the fluids, liquids necessary for amplifying, fragmentizing, ligation-causing or labeling SNP and genome DNA for a variation analysis, and liquids necessary for reverse-transcribing cDNA from RNA and amplifying, fragmentizing, ligation-causing or labeling the cDNA.
The fluid containing cartridge according to any one of Claims 1 to 13, wherein the container portions contain, as the fluids, liquids necessary for amplifying, fragmentizing or labeling genome DNA for a chromosome anomaly analysis.
The fluid containing cartridge according to any one of Claims 1 to 15, wherein the atmosphere flowing portion comprises an atmosphere flowing passage having an atmosphere hole and communicating at least one of the container portions with the outside, and a porous material disposed in the atmosphere flowing passage and allowing passing of the atmosphere, but not allowing passing of the fluid.
The fluid containing cartridge according to Claim 16, wherein the atmosphere flowing portion is connected to two or more of the container portions.
A fluid reaction unit comprising:
the fluid containing cartridge according to any one of Claims 1 to 17; and

a reaction vessel being connectable to the plurality of connecting and communicating portions disposed in the fluid containing cartridge and being able to contain the fluid supplied through the connecting and communicating portion connected.
The fluid reaction unit according to Claim 18, wherein the reaction vessel is formed in the shape of a tube gradually narrowing toward the connecting and communicating portion.
A reaction apparatus for mixing a plurality of fluids with each other to develop a reaction, the reaction apparatus comprising:
a mounting unit capable of mounting the fluid containing cartridge according to any one of Claims 1 to 17;

a reaction vessel being connectable to the plurality of connecting and communicating portions disposed in the mounted fluid containing cartridge and being able to contain the fluid supplied through the connecting and communicating portion connected;

a moving unit for moving at least one of the reaction vessel and the mounted fluid containing cartridge to a predetermined connecting position where the reaction vessel and any one of the plurality of connecting and communicating portions are connected to each other; and

a pressure applying unit capable of applying differential pressure to act upon the container portion of the fluid containing cartridge, thereby supplying the fluid contained in the container portion to the reaction vessel.
The reaction apparatus according to Claim 20, further comprising a control unit for, in accordance with a series of reaction procedures to be executed by using the fluids contained in the container portions of the mounted fluid containing cartridge, controlling the moving unit such that one of the plurality of container portions is selectively connected in turn to the reaction vessel, and controlling the pressure applying unit to transfer the fluid by the action of the differential pressure applied to the container portion.
The reaction apparatus according to Claim 20 or 21, wherein the pressure applying unit is connected to the reaction vessel to suck the fluid from the container portion into the reaction vessel by lowering air pressure in the reaction vessel and to push out the fluid from the reaction vessel to the container portion by raising air pressure in the reaction vessel.
The reaction apparatus according to any one of Claims 20 to 22, wherein the mounting unit mounts the fluid containing cartridge provided with a circular disk-shaped housing in which the container portions are formed, and
wherein the moving unit moves at least one of the reaction vessel and the mounted fluid containing cartridge by rotationally moving one of the reaction vessel and the fluid containing cartridge about an axis of rotation.
The reaction apparatus according to Claim 23, wherein the mounting unit mounts the fluid containing cartridge by using a contact member disposed on a flat surface of the housing having the shape of a circular disk, in which the connecting and communicating portions are formed, and further disposed in a circular shape coaxial with the axis of rotation about which the housing is rotationally moved, the contact member contacting with the flat surface of the fluid containing cartridge and having a through-hole communicating with the connecting and communicating portion, and by using a fixing member for rotatably fixing the fluid containing cartridge in a state of the contact member being held in contact with the flat surface of the fluid containing cartridge, and
wherein the reaction vessel is connected to the through-hole formed in the contact member.
The reaction apparatus according to any one of Claims 20 to 24, further comprising a cartridge temperature adjusting unit capable of adjusting temperature of the fluid containing cartridge mounted to the mounting unit; and
a reaction-vessel temperature adjusting unit capable of adjusting temperature of the reaction vessel.
A reaction method using a reaction apparatus for mixing a plurality of fluids with each other to develop a reaction, the reaction method comprising the steps of:
(a) preparing the fluid containing cartridge according to any one of Claims 1 to 17, which is mounted to the reaction apparatus, and a reaction vessel which is connectable to the plurality of connecting and communicating portions disposed in the mounted fluid containing cartridge and which is able to contain the fluid supplied through the connecting and communicating portion connected to the reaction vessel, and moving at least one of the reaction vessel and the mounted fluid containing cartridge to a predetermined connecting position where the reaction vessel and any one of the plurality of connecting and communicating portions are connected to each other; and

(b) applying pressure to the container portion of the fluid containing cartridge to introduce the fluid contained in the relevant container portion to the reaction vessel.
A gene analysis method of detecting SNP, variation of DNA, or representation of RNA by using the fluid containing cartridge according to Claim 14.
A gene analysis method of measuring change in the number of chromosome copies for a human chromosome by using the fluid containing cartridge according to Claim 15.