WO2010001636A1 - Puce à adn - Google Patents

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Publication number
WO2010001636A1
WO2010001636A1 PCT/JP2009/053576 JP2009053576W WO2010001636A1 WO 2010001636 A1 WO2010001636 A1 WO 2010001636A1 JP 2009053576 W JP2009053576 W JP 2009053576W WO 2010001636 A1 WO2010001636 A1 WO 2010001636A1
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WO
WIPO (PCT)
Prior art keywords
reaction
probe
dna
width
reaction channel
Prior art date
Application number
PCT/JP2009/053576
Other languages
English (en)
Japanese (ja)
Inventor
真寛 村里
和成 山田
晃暢 織部
Original Assignee
日本碍子株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 日本碍子株式会社 filed Critical 日本碍子株式会社
Priority to JP2010518944A priority Critical patent/JPWO2010001636A1/ja
Publication of WO2010001636A1 publication Critical patent/WO2010001636A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00421—Means for dispensing and evacuation of reagents using centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00495—Means for heating or cooling the reaction vessels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B01J2219/00531—Sheets essentially square
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B01J2219/00536—Sheets in the shape of disks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0636—Focussing flows, e.g. to laminate flows
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0874—Three dimensional network
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions

Definitions

  • the present invention relates to a DNA array.
  • a DNA array having a flow path in which a plurality of DNA spots are formed is known.
  • the DNA array described in Patent Document 1 has a plurality of DNA spots in which a plurality of DNA spots are formed and a plurality of capillary structures (reaction channels) having the same diameter as the solution inlet (inlet) and solution outlet (outlet).
  • the DNA array is used so as to cause a hybridization reaction between the target DNA and the probe DNA formed in the reaction channel by flowing a solution containing the target DNA into the reaction channel.
  • the width of the reaction channel is made equal to the width of the inlet in the portion close to the inlet, and gradually widened as the area where the probe DNA is formed. It is conceivable that the gap is almost constant.
  • the flow rate of the solution in this region may be biased depending on the location.
  • the present invention has been made in view of the above-described problems, and the difference in the chances of a hybridization reaction between a plurality of types of probe DNAs formed in the width direction of the reaction channel and the target DNA flowing in the reaction channel.
  • the main object is to provide a DNA array that can be suppressed.
  • the present invention employs the following means in order to achieve the above-described object.
  • the first DNA array of the present invention comprises: A reaction channel for flowing a solution containing the target DNA from the inlet toward the outlet; A first probe formation region provided in the middle of the reaction channel so that the width is substantially constant, and a plurality of types of probe DNAs formed at least along the width direction of the reaction channel; A DNA array comprising: The width of the reaction channel is formed so as to gradually widen from the inlet toward the first probe formation region, The depth of the reaction channel is shallower as the width of the reaction channel is wider and deeper as the width of the reaction channel is narrower.
  • the width of the reaction channel is formed so as to gradually widen from the inlet toward the first probe formation region, and the depth of the reaction channel becomes shallower as the width of the reaction channel becomes wider.
  • the reaction channel is formed deeper as the width is narrower. For this reason, the change in the cross-sectional area of the reaction flow path from the inlet to the first probe formation region is smaller than that in the case where the depth is constant regardless of the width, and the location within the first probe formation region is small. The difference in the flow rate of the solution due to the difference is suppressed to a small level.
  • the probe formation region after the hybridization reaction can be washed relatively evenly, and as a result, the whole can be washed with a small amount of washing solution.
  • the reaction channel may have a substantially constant cross-sectional area from the inlet to the outlet.
  • the first DNA array of the present invention may be provided with a first groove that is a groove along the reaction channel provided on both sides in the width direction of the first probe formation region.
  • the first DNA array of the present invention is provided with a plurality of types of probe DNAs formed along at least the width direction of the reaction flow path so as to have a substantially constant width downstream of the first probe formation region.
  • the reaction channel has a U-shape in which both ends are connected to the inlet and the outlet, and the width of the curved portion is narrower than the straight portion, and the first probe is formed.
  • the region is provided in a straight portion between the curved portion of the reaction channel and the inlet, and the second probe formation region is provided in a straight portion between the curved portion of the reaction channel and the outlet. It is good as it is.
  • the width of the reaction channel may be formed so as to gradually narrow from the second probe formation region toward the outlet. If it carries out like this, it will become possible to discharge
  • the first DNA array of the present invention in which the reaction channel has a U shape is a groove in the direction along the reaction channel provided on both sides in the width direction of the first probe formation region.
  • a groove and a second groove that is a groove along the reaction flow path provided on both sides in the width direction of the second probe formation region may be provided.
  • the second DNA array of the present invention comprises: A reaction channel for flowing a solution containing the target DNA from the inlet toward the outlet; A probe formation region provided in the middle of the reaction channel, wherein a plurality of types of probe DNAs are formed along the width direction of the reaction channel with at least a substantially constant width; A DNA array comprising: Grooves in the direction along the reaction flow path are provided on both sides of the probe formation region in the width direction.
  • grooves in the direction along the reaction flow path are provided on both sides in the width direction of the probe formation region, and when the solution passes through the probe formation region, the groove flows along the solution flowing through the groove. Since the solution flows through the probe formation region, it is possible to suppress a decrease in the flow rate of the solution flowing through the end portion of the probe formation region. For this reason, compared with the case where there is no groove, the difference in the flow rate of the solution due to the difference in the location of the probe formation region can be suppressed. Therefore, it is possible to suppress a difference in the chance of performing a hybridization reaction with the target DNA flowing in the reaction channel between a plurality of types of probe DNAs formed in the width direction of the reaction channel.
  • the first and second DNA arrays of the present invention include a supply port that is connected to the inlet and supplies liquid from the inlet, a waste liquid tank that stores waste liquid, and a waste liquid that is connected to the outlet and discharged from the outlet.
  • a solution containing instrument having a discharge port for receiving into a tank is mounted, and a solution containing the target DNA is supplied from the inlet via the supply port, so that the probe DNA in the probe formation region and the target DNA are hybridized.
  • the waste liquid after hybridization reaction is stored in the waste liquid tank through the outlet, the supplied liquid is switched to the cleaning liquid, and the cleaning liquid is supplied from the inlet through the supply port.
  • the waste liquid of the cleaning liquid is stored in the waste liquid tank through the outlet. Or as used in the so that. In this way, a series of processes of the hybridization reaction between the probe DNA and the target DNA and the cleaning of the probe formation region can be performed relatively easily with one solution storage device.
  • FIG. Explanatory drawing of the cross section after cartridge mounting mechanism 80 and cartridge mounting. Explanatory drawing of the deaeration groove
  • FIG. FIG.
  • FIG. 4 is a partial cross-sectional view of the cartridge 350 taken along the line B-B ′.
  • Explanatory drawing of the heat insulation structure of the reaction apparatus 90 FIG.
  • Explanatory drawing of the procedure when amplifying and adjusting rice genomic DNA Explanatory drawing of the procedure which makes adjusted DNA react with probe DNA.
  • FIG. 1 is a configuration diagram showing an outline of the configuration of the reaction device 90
  • FIG. 2 is an explanatory diagram of the cartridge mounting mechanism 80
  • FIG. 2 (a) is an exploded perspective view
  • FIG. 2 (b) is a perspective view
  • FIG. 2C is a cross-sectional view taken along the line AA ′ of FIG. 2B
  • FIG. 3 is an explanatory view of a deaeration groove provided in the reaction tank 30
  • FIG. 4 is an external view of the cartridge 350
  • FIG. 6 is an explanatory diagram of the second layer 351b of the cartridge 350
  • FIG. 7 is an explanatory diagram of the third layer 351c of the cartridge 350
  • FIG. 8 is an explanatory diagram of the fourth layer 351d of the cartridge 350
  • FIG. I is an external view of the mini-array 350b
  • FIG. 10 is an explanatory diagram of the mini-array 350b
  • FIG. 11 is a cross-sectional view taken along the line CC ′ of the mini-array 350b of FIG. It is explanatory drawing of a heat insulation structure.
  • the liquid storage portions 302 to 304, 308, 309, 311, 315 to 321, 323, and 325 and the waste liquid tanks 327 and 328, which can store liquids are collectively referred to as “chamber”. It shall be.
  • the reaction device 90 will be described as identifying rice varieties from DNA.
  • the reaction device 90 includes a cartridge mounting mechanism 80 capable of mounting a cartridge 350 having a guide portion 352 on the upper surface, which is stored in individual liquid storage portions so that a plurality of types of reagents (liquids) can be taken out.
  • the reaction tank 30 that can be connected to a plurality of ports arranged in the mounted cartridge 350 and can store the liquid supplied from the connected port, and the reaction tank 30 that connects any port of the cartridge 350
  • the rotation mechanism 32 that rotates and moves the cartridge 350 around its central axis to the position of the cartridge 350, and the liquid stored in the liquid storage section can be supplied to the reaction tank 30 by applying a differential pressure to the liquid storage section of the cartridge 350.
  • the reaction apparatus 90 includes a rectangular base 90a disposed at the lowermost part thereof, and a side L-shaped support member 92 disposed on the front side of the base 90a.
  • the support member 92 is formed with a middle step surface 92a and an upright wall portion 92b that is erected upward on the back side of the middle step surface 92a. Further, the pump 34, the controller 40, and the like are disposed on the back side of the support member 92.
  • the cartridge mounting mechanism 80 includes a rotating disk 82 into which the reaction tank 30 is inserted and fitted into the guide portion 352 of the cartridge 350, a presser 84 that urges the rotating disk 82 downward from above, and a cartridge.
  • Rotating stage 38 (see FIG. 1) on which 350 is mounted.
  • a fluorine-based material for example, Teflon (registered trademark, the same applies hereinafter)
  • Teflon a fluorine-based material
  • Teflon a fluorine-based material
  • the rotating disk 82 is packed on the contact surface 352a on the inner peripheral side of the guide portion 352 of the cartridge 350 in which a plurality of ports are arranged in a circle.
  • One flow passage 82a is provided which is in contact with each other via 354 and allows any one of the ports to communicate with the reaction tank 30.
  • the packing 354 is integrally formed into a shape in which a plurality of O-rings corresponding to each port are connected.
  • the rotating disk 82 is rotatably fitted in the guide portion 352 of the cartridge 350, and any one of the ports (that is, any one chamber) and the reaction tank 30 through the arranged flow path 82a. To communicate with each other.
  • the rotating disk 82 is inserted into the guide portion 352, blocks the flow of the outside air from other ports not communicating with the flow passage 82a, and transfers the liquid only from the port connected to the flow passage 82a.
  • the presser 84 is a member that urges the rotary disk 82 downward from above so that the cartridge 350 can rotate while the rotary disk 82 is in contact with the contact surface 352a of the cartridge 350 placed on the rotary stage 38. It is.
  • the presser 84 is biased downward from above so as to sandwich the two stepped portions 82b on the upper surface of the rotary disk 82, thereby restricting the vertical movement and rotational movement of the cartridge 350, thereby rotating the cartridge 350. Even in this case, the absolute position of the flow passage 82a of the rotary disk 82 is maintained at the same position. As a result, by rotating the cartridge 350, only one of the ports can communicate with the reaction tank 30.
  • the rotation mechanism 32 includes a rotation stage 38 on which the cartridge 350 is placed, and a motor 37 that rotates the rotation stage 38 stepwise so as to be fixed at a predetermined connection position.
  • the rotary stage 38 has a disk shape, and is rotatably supported on the middle surface 92 a of the support member 92.
  • the rotary stage 38 is made of copper electrolessly plated with nickel, and a plurality of projections (not shown) are formed at irregular positions on the upper surface.
  • a plurality of concave portions (grooves 342 (see FIG. 8)) into which the convex portions enter are formed on the bottom surface of the cartridge 350 at positions corresponding to the arrangement of the convex portions, and the convex portions and the concave portions are fitted.
  • the cartridge 350 When the cartridge 350 is placed on the rotary stage 38 in the state, the cartridge 350 can be fixed at a predetermined initial connection position.
  • a cartridge Peltier element 38 a capable of adjusting the temperature of the cartridge 350 is disposed inside the rotary stage 38. By adjusting the temperature of the rotary stage 38 by the cartridge Peltier element 38a, the mounted cartridge 350 can be adjusted to a constant temperature.
  • an alumite-treated aluminum may be used as a material of the rotary stage 38.
  • the reaction tank fixing part 36 is made of copper electrolessly plated with nickel, and is fixed to the center of the standing wall part 92 b of the support member 92, and above the cartridge 350 placed on the rotary stage 38. Is detachably fixed.
  • the reaction tank fixing part 36 includes a reaction tank Peltier element 36a capable of adjusting the temperature of the reaction tank 30 inside.
  • the reaction tank 30 can be adjusted to a constant temperature by adjusting the temperature of the reaction tank fixing portion 36 by the reaction tank Peltier element 36a.
  • fixed part 36 you may use what alumite-treated aluminum.
  • the reaction vessel 30 is a member made of polypropylene, and is a tube-shaped member that becomes thinner toward the lower side, that is, closer to the port, as shown in FIGS.
  • the reaction tank 30 has a rotating disk 82 attached to the lower end via an O-ring 54b (see FIG. 2C), and an air supply / exhaust tube 34a connected to the upper end as shown in FIG.
  • the pressure generated by the operation of the pump 34 acts on the reaction tank 30 via the air supply / exhaust tube 34 a, and the pressure acts on any chamber of the cartridge 350 connected via the rotary disk 82.
  • the reaction tank 30 stores liquid sucked from the liquid storage portions 302 to 304, 308, 309, 311, 315 to 321, 323, and 325, agitates the stored liquid, and various types of liquids depending on the stored liquid.
  • the reaction of Further, as shown in FIG. 3, a deaeration groove 30 a that is a vertical groove is provided inside the reaction tank 30.
  • the pump 34 is a so-called tube pump that applies pressure to the tip connected to the tube by squeezing the tube with a roller. As shown in FIG. 1, the pump 34 is connected to the air supply / exhaust tube 34 a and applies pressure to the liquid stored in the chamber of the cartridge 350 via the air supply / exhaust tube 34 a and the reaction tank 30. In addition, the pump 34 sets the rotation direction, the number of steps (the number of rotations), and the speed of the stepping motor connected to the pump 34 to increase or decrease the pressure applied to the tip connected to the tube. You can do it.
  • a stepping motor connected to the pump 34 is used to switch between the operation of storing liquid from the reaction tank 30 to the cartridge 350 side and the operation of supplying liquid from the cartridge 350 side to the reaction tank 30.
  • the rotation direction, the number of steps (number of rotations), and the speed are set, and then the pump 34 is operated. Further, when it is necessary to adjust the pressure acting on the tip connected to the tube, the rotation direction of the stepping motor and the number of steps so that the output value of a pressure gauge (not shown) installed in the air supply / exhaust tube 34a indicates the target pressure. (Rotation speed) and speed shall be set.
  • the controller 40 is configured as a microprocessor centered on the CPU 42, and includes a flash ROM 43 that stores various processing programs, and a RAM 44 that temporarily stores data and saves data. From the controller 40, a control signal including a signal for changing the connection state of the pump 34 to the supply / exhaust tube 34a, a control signal to the motor 37, a supply voltage to the Peltier element 36a for the reaction tank and the Peltier element 38a for the cartridge, etc. Is output.
  • the cartridge 350 includes a cartridge main body 350a that stores various liquids for identifying rice varieties, and a miniarray 350b that is detachably attached to the main body.
  • the cartridge main body 350a is a member made of a cycloolefin copolymer, and is composed of four layers of a first layer 351a to a fourth layer 351d formed in a disc shape. On the upper surface of the first layer 351a, As shown in FIG. 5, a guide portion 352 into which the rotary disk 82 (see FIG. 2) is fitted is provided.
  • the cartridge main body 350a includes three grooves 342 extending in the radial direction on the lower surface of the fourth layer 351d and a filling hole 341 for filling the column, and an O-ring is installed in the third layer 351c and the fourth layer 351d. Auxiliary holes 340c and 340d.
  • the cartridge main body 350a includes a plurality of liquid storage portions 302 to 304, 308, 309, 311, 315 to 321 and 323 that can store a liquid in a predetermined volume determined by the liquid to be stored. , 325, and circulation ports 302a to 304a, 308a, each of which is disposed at a predetermined connection position that connects and connects any one of the liquid storage units to the reaction tank 30 by rotating and switching the cartridge 350.
  • liquid storage portions 302 to 304, 308, 309, 311, 315 to 321, 323, and 325 communicate with the outside air and liquid storage portions 302 to 304, 308, 309, 311, 315 to 321, 323, 325 takes outside air or liquid storage portions 302 to 304 308, 309, 311, 315 to 321, 323, 325, the outside air circulation unit 326, the waste liquid tanks 327 and 328 that can store the waste liquid supplied from the reaction tank 30, and the reaction produced in the reaction tank 30
  • the liquid storage portions 302 to 304, 308, 309, 311, 315 to 321, 323, and 325 are spaces formed in a shape in which both ends are narrowed.
  • the liquid storage portions 304, 308, 309, 315, 316, 318, 319, 321, and 323 having a large amount of liquid to be stored are one over the second layer 351b and the third layer 351c.
  • the liquid storage portions 302, 303, 311, 317, 320, and 325 formed as spaces and containing a small amount of liquid are formed only in one of the second layer 351b and the third layer 351c.
  • one end of the liquid storage portions 302 to 304, 308, 309, 311, 315, 316, 318, 319, 321, 323, and 325 near the center of the cartridge 350 is formed on the lower surface of the third layer 351c.
  • the flow paths 302b to 304b, 308b, 309b, 311b, 315b, 316b, 318b, 319b, 321b, 323b, and 325b, and the third layer 351c and the second layer 351b are connected to the side surface near the bottom of the liquid storage unit.
  • the flow ports are connected to the distribution ports 302a to 304a, 308a, 309a, 311a, 315a, 316a, 318a, 319a, 321a, 323a, and 325a, respectively, through the directional flow paths.
  • One end of the liquid container 317, 320 near the center of the cartridge 350 is formed on the lower surface of the second layer 351b and connected to the side surface near the bottom of each liquid container 317b, 320b, and the second layer. It is connected to the distribution ports 317a and 320a via the vertical flow path 351b.
  • the distribution ports 302a to 304a, 308a, 309a, 311a, 315a to 321a, 323a, and 325a communicate with the liquid storage units 302 to 304, 308, 309, 311, 315 to 321, 323, and 325, respectively, and the liquid storage unit 302 304, 308, 309, 311, 315 to 321, 323, 325 are holes used for supplying liquid, and are provided on the inner peripheral side (contact surface 352a) of the first layer 351a.
  • the circulation ports 302a to 304a, 308a, 309a, 311a, 315a to 321a, 323a, and 325a are respectively arranged on the same rotation axis where the cartridge 350 is rotated by the rotation mechanism 32, that is, on the same circular plane coaxial with the central axis of the cartridge 350. Is arranged. Further, the liquid storage portions 302 to 304, 304, 304, 308, 309, 311, 315 to 321, 323, and 325 connected to the flow ports are subjected to a differential pressure acting on the liquid. Liquids stored in 308, 309, 311, 315 to 321, 323, 325 can be supplied to the reaction tank 30.
  • the outside air circulation part 326 is formed on the lower surface of the third layer 351c, and the outside air passages 302c, 303c, 309c extending radially outward from one end of the liquid storage parts 302, 303, 309, 311, 325 far from the center of the cartridge.
  • 311c, 325c, the outer airflow passages 317c, 320c formed on the lower surface of the second layer 351b and extending radially outward from one end of the liquid container 317, 320 far from the center of the cartridge, and the first layer 351a Ventilation holes 302d to 304d, 308d, 309d, 311d, 315d to 321d, 323d, and 325d formed in the direction are generic names.
  • vent holes 302d to 304d, 308d, 309d, 311d, 315d to 321d, 323d, and 325d are external air flow paths 302c, 303c, 309c, 311c, and 325c, respectively.
  • the liquid storage units 302, 303, 309, 311 and 325 are communicated with the outside air through flow paths formed in the vertical direction of the second layer 351b and the third layer 351c.
  • the vent holes 317d and 320d directly connect the liquid storage portions 317 and 320 to the outside air through channels formed in the vertical direction in the external airflow passages 317c and 320c and the second layer 351b, respectively.
  • the air holes 304d, 308d, 315d, 316d, 318d, 319d, 321d, and 323d are connected to the liquid storage portions 304, 308, 315, 316, 318, 319, 321 and 323 without passing through the flow path. Is to communicate.
  • the outside air circulation part 326 may be provided with a hydrophobic porous material that does not allow liquid to pass but allows outside air to pass therethrough.
  • the hydrophobic porous material for example, a Teflon porous material (Temish manufactured by Nitto Denko Corporation) can be used.
  • the waste liquid tanks 327 and 328 are spaces provided on the outermost periphery of the cartridge 350 and are formed as one space extending between the second layer 351b and the third layer 351c.
  • the waste liquid tank 327 is connected to the waste liquid tank 327 and formed in the second layer 351b, extending in the radial direction, and a second layer from one end of the waste liquid flow path 327e closer to the center of the cartridge 350. It is connected to the column section 306 via a flow path that penetrates 351b in the vertical direction and a diffusion flow path 327f that extends in the radial direction and is connected to the flow path.
  • the fluid that has passed through the column portion 306 from the combined circulation port 306 a is discharged to the waste liquid tank 327.
  • the width of the diffusion channel 327 f is substantially the same as the width of the column portion 306.
  • the waste liquid tank 328 is connected to a vertical flow path 328f provided in the second layer 351b via a waste liquid flow path 328e connected to the waste liquid tank 328.
  • the first layer 351a is provided with vent holes 327d and 328d that allow the waste liquid tanks 327 and 328 to communicate with the outside air.
  • a water-absorbing porous material that absorbs the waste liquid may be disposed inside the waste liquid tanks 327 and 328 so that the waste liquid that has once flowed in is reliably held in the waste liquid tanks 327 and 328.
  • a sponge or the like can be used as the water-absorbing porous material.
  • the column unit 306 is provided between the combined flow port 306a and the diffusion flow path 327f and includes a column.
  • a ceramic column such as silica gel
  • the pump 34 is operated to pressurize the inside of the reaction tank 30 to circulate the liquid stored in the reaction tank 30 through the column portion 306 and store it in the diffusion flow path 327f, if further pressurized, the diffusion flow path 327f
  • the liquid stored in is stored in the waste liquid tank 327, and when the pressure is reduced, the liquid passes through the column portion 306 and is stored in the reaction tank 30 again.
  • the column portion 306 is filled with the column by filling the column from the lower surface side of the fourth layer 351d through the filling hole 341 and then covering the lower surface of the fourth layer 351d.
  • the joint flow ports 306a and 313a are holes that communicate with the waste liquid tanks 327 and 328, respectively, and are used when the liquid is stored in the waste liquid tanks 327 and 328, and are connected to the inner peripheral side (contact surface 352a) of the first layer 351a. ).
  • the coupling flow ports 306a and 313a are respectively disposed on the same rotational axis on which the cartridge 350 is rotated by the rotation mechanism 32 (see FIG. 1), that is, on the same circular plane coaxial 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 that are not perforated, and their positions are defined by the packing 354. These closed ports close the lower surface of the reaction tank 30 when positioned in a state facing the reaction tank 30, so that the liquid sucked into the reaction tank 30 can be pressurized by the pressurizing operation or the depressurizing operation of the pump 34. It is possible to reduce the pressure.
  • the closed flow path 310 is formed as a single groove in the third layer 351c, and the flow path 310b extending in the radial direction formed in the third layer 351c, and the vertical direction formed in the third layer 351c and the second layer 351b. Are connected to the injection port 310a.
  • One end of the closed channel 310 on the side away from the center of the cartridge 350 is not connected to the outside air circulation unit 326, unlike the liquid storage unit described above. For this reason, when the closed flow path 310 and the reaction tank 30 are not in communication, the lower surface of the rotating disk 82 closes the injection port 310a, and the closed flow path 310 becomes a sealed space.
  • the injection port 310a is a hole that communicates with the closed channel 310 and is used when the liquid is stored in the closed channel 310 or the liquid stored in the closed channel 310 is supplied to the reaction tank 30. It is provided on the inner peripheral side (contact surface 352a) of the layer 351a.
  • the injection port 310 a is arranged together with other ports on the same circular plane that is coaxial with the rotation axis around which the cartridge 350 is rotated by the rotation mechanism 32, that is, the central axis of the cartridge 350.
  • the mini-array 350b is detachably formed in a slot 330 formed by the third layer 351c and the fourth layer 351d of the cartridge body 350a. .
  • the handle 367 is provided at the base end portion of the mini-array 350b, the mini-array 350b can be easily inserted into or removed from the slot 330.
  • the mini-array 350b has first and second connection ports 361 and 362 provided side by side on the tip side, and both ends connected to the first and second connection ports 361 and 362.
  • the first and second grooves 381a and 381b provided on both sides of each of the 366b and a step portion 371 provided on the side surface of the mini-array 350b are provided.
  • an inclined portion 368 is provided on the front end side of the mini array 350b, and a tapered portion 369b that is tapered toward the front end is provided on the mini array 350b.
  • the tapered portion 369b contacts the tapered portion 369a (see FIG. 7) formed in the slot 330 at an angle corresponding to the tapered portion 369b. By making contact, the mini-array 350b is reliably positioned.
  • the first connection port 361 is connected to the longitudinal flow path 328g formed in the second layer 351b and communicating with the combined flow port 313a, as shown in FIG. It is connected via a packing 355 having a shape in which two O-rings are connected. Further, the second connection port 362 is connected via a packing 355 to a vertical flow path 328f (see also FIG. 6) formed in the second layer 351b.
  • FIG. 6 A cross-sectional view of the slot 330 before the mini-array 350b is inserted into the slot 330 is shown in FIG.
  • the packing 355 does not catch on the mini array 350b because the inclined portion 368 provided in the mini array 350b first hits.
  • the packing 355 is attached from the lower surface side of the fourth layer 351d through auxiliary holes 340c (see also FIG. 7) and 340d (see also FIG. 8) provided in the third layer 351c and the fourth layer 351d, respectively. Is done.
  • the first and second spot areas 366a and 366b of the mini-array 350b are hybridized when the DNA solution flows from the reaction tank 30 through the first connection port 361, respectively.
  • a plurality of probe DNAs are formed. Specifically, for example, in the first spot area 366a, a plurality of probe DNAs are arranged at regular intervals in the width direction of the reaction channel 365, and a plurality of probe DNAs are arranged at regular intervals in the length direction. This also applies to the second spot area 366b.
  • the mini-array 350b includes a lower member 363 and an upper member 364, which are made of an adhesive sheet 370 (for example, 531N # 80 (manufactured by Nitto Denko Corporation) or a titer stick (Kazix). And made by bonding)).
  • the lower member 363 is a plate-shaped member made of polycarbonate and having a thickness of 0.3 mm.
  • the upper member 364 is a plate-like member made of polycarbonate and having a thickness of 1 mm, and a groove corresponding to the reaction channel 365 is formed on the back surface.
  • the adhesive sheet 370 is also formed with a U-shaped through hole having a shape corresponding to the reaction channel 365.
  • the reaction channel 365 is formed by superposing and bonding the upper member 364, the lower member 363, and the adhesive sheet 370.
  • the lower member 363 having a small thickness is in contact with the four layers 351d of the cartridge main body 350a, and the fourth layer 351d is in contact with the rotary stage 38 (see FIG. 1).
  • the temperature of the liquid in the reaction channel 365 can be easily adjusted by the cartridge Peltier element 38 a in the rotary stage 38.
  • the probe DNAs in the first and second spot areas 366a and 366b are formed on the lower surface of the upper member 364 (the surface on the side where the reaction channel 365 is formed).
  • the reaction channel 365 gradually increases in width and gradually decreases in depth from the first connection port 361 toward the first spot area 366a. Is formed. Further, the width is gradually reduced from the second spot area 366b toward the second connection port 362, and the depth is gradually increased. Furthermore, the width of the U-shaped curved portion 365a is narrower than the straight portion 365b, and is formed so that the depth gradually increases from the straight portion 365b toward the curved portion 365a.
  • the reaction flow path 365 has a width and depth so that the cross-sectional area is constant from the first connection port 361 to the second connection port 362, and the width and depth of the first and second grooves 381a and 381b. Is set.
  • the first and second grooves 381a and 381b are provided in the upper member 364, and the adjacent portions in the width direction of both spot areas 366a and 366b of the reaction channel 365 are The first and second grooves 381a and 381b are deeper than the first and second spot areas 366a and 366b. Further, the width and depth of the reaction channel 365 are formed by digging a groove in the upper member 364 in addition to the shape and thickness of the adhesive sheet 370 except for the straight portion 365b. About the linear part 365b of the reaction flow path 365, the width and depth are formed by the shape and thickness of the adhesive sheet 370.
  • a cartridge 350 in which a mini array 350b is inserted into a container body 350a in advance is used.
  • the cartridge 350 stores liquids including reagents used for a predetermined reaction in the liquid storage portions of the desired amount cartridge 350, and the liquid storage portions 302 to 304, 308, 309, 311, 315 to 321, 323, respectively.
  • the cartridge 37 is moved by the motor 37. The position of the port of the cartridge 350 connected to the reaction vessel 30 by sequentially rotating is changed as appropriate.
  • the reaction product when purifying a reaction product, the reaction product is adsorbed on the column to discharge unnecessary liquid to the waste liquid tank 327, or the reaction product adsorbed on the column is stored in one of the liquid storage units. It is carried out by elution with the liquid thus prepared and once stored in the diffusion channel 327 f and then supplied to the reaction tank 30. Further, as shown in FIG. 13, in the reaction apparatus 90, since the reaction tank 30 is provided outside the cartridge 350, the temperature change of the reaction tank 30 is not easily transmitted to the cartridge 350, and the reaction tank 30 and the cartridge 350 are not transmitted. Can be held at different temperatures (for example, reaction temperature, storage temperature, etc.).
  • a motor 72 capable of rotating a magnet 70 is provided beside the reaction tank fixing portion 36, and a rotor 74 including a magnet is placed in the reaction tank 30, and is rotated by rotating the magnet 70 by the motor 72.
  • the liquid in the reaction tank 30 can be stirred by rotating the child 74.
  • a neodymium magnet may be used as the magnet and the magnet 70 included in the rotor 74.
  • FIG. 14 is an explanatory diagram of the procedure for amplifying and adjusting rice genomic DNA
  • FIG. 15 shows the reaction of the adjusted genomic DNA with the probe DNA formed in the first and second spot areas 366a and 366b of the miniarray 350b. It is explanatory drawing of the procedure made to do.
  • the liquid storage portion and waste liquid tanks 327 and 328 of the cartridge 350, the connected distribution port and injection port, and the reaction tank 30 are schematically shown.
  • the liquid storage units 302 to 304, 308, 309, 311, 315 to 321, 323, 325 and the waste liquid tanks 327 and 328 are described in FIG. 5 to FIG.
  • symbol of each structure made is described.
  • a blank chamber indicates that no liquid is contained therein.
  • the reaction tank 30 is represented by an ellipse when a liquid is contained in the reaction tank 30, and is represented by a rectangle when processing is performed on the contained liquid. When not contained, it is represented by a blank ellipse.
  • the arrow in a figure represents the direction through which a liquid or gas flows.
  • step numbers are given to the reaction tank 30 portions.
  • genomic DNA amplification / adjustment processing will be described with reference to FIGS.
  • the user puts rice genomic DNA, whose varieties are to be identified, into the reaction tank 30 and connects it to the rotating disk 82, and inserts it into the cartridge 350 to form a reaction unit (see FIG. 2B). Then, a door (not shown) provided on the side surface of the reaction vessel fixing portion 36 is opened, and the upper portion of the reaction vessel 30 communicates with the air supply / exhaust tube 34a and is slid from the side surface so that the rotating disk 82 is urged downward by the presser 84. The lever reaction unit is placed on the rotary stage 38.
  • the presser 84 since the presser 84 is bent because the material is Teflon, the presser 84 is placed so that the plurality of concave portions formed on the bottom surface of the cartridge 350 fit into the plurality of convex portions provided on the upper surface of the rotary stage 38. And mounted in a state of being biased downward by the presser 84. Then, a start button (not shown) is pressed. Then, the CPU 42 of the controller 40 reads and executes the DNA adjustment processing routine stored in the flash ROM 43. When this routine is executed, the CPU 42 first drives the motor 37 to rotate the cartridge 350 so that the flow port 302a communicates with the reaction tank 30 and operates the pump 34 to lower the pressure in the reaction tank 30. Then, the liquid stored in the liquid storage unit 302 is sucked into the reaction tank 30 (step S1100).
  • step S1110 the flow port 303a and the reaction tank 30 are communicated, and the pump 34 is operated to suck out the liquid stored in the liquid storage unit 303 (step S1110).
  • the cartridge 350 is rotated so that the closed port 305a and the reaction vessel 30 are connected, and the reaction vessel 30 is stirred for 15 minutes while maintaining the temperature in the reaction vessel 30 at 95 ° C.
  • the temperature in the reaction vessel 30 is changed.
  • a cycle of stirring at 95 ° C. for 1 minute, stirring at a temperature of 66 ° C. for 1 minute and 30 seconds, and stirring at a temperature of 72 ° C. for 30 seconds is repeated 40 cycles, and finally stirring is performed at a temperature of 72 ° C. for 10 minutes to react (step S1120). .
  • “stirring” refers to mixing the solution in the reaction tank 30 by rotating the rotor 47 placed in the reaction tank 30 by the motor 72.
  • the flow port 304a is communicated with the reaction tank 30, and the pump 34 is operated to suck out the liquid (adsorption buffer (3.8 mol / L ammonium sulfate)) stored in the liquid storage unit 304 (step S1130).
  • the combined flow port 306a and the reaction tank 30 are communicated, and the pump 34 is operated to flow the mixed solution in the reaction tank 30 to the column unit 306 (step S1140).
  • the mixed solution flows in through the column flow port 306 a of the first layer 351 a of the cartridge 350 shown in FIG.
  • the flow port 323a and the reaction tank 30 are communicated, the pump 34 is operated, and the liquid (first cleaning buffer (1.9 mol / L ammonium sulfate)) stored in the liquid storage unit 323 is sucked out.
  • the temperature inside is maintained at 25 ° C. and stirred for 1 minute to wash the inside of the reaction vessel 30 (step S1150).
  • the reason for washing the inside of the reaction vessel 30 is to prevent the salt from being precipitated.
  • the pump 34 is operated to store the cleaned liquid in the reaction tank 30 in the liquid storage unit 323 (step S1160).
  • step S1170 the liquid (second washing buffer (pH 6.0, 10 mmol / L, phosphoric acid-ethanol mixed solution (mixed) The ratio 1: 2.8))) is sucked out.
  • step S1180 the pump 34 is operated to flow the second washing buffer in the reaction tank 30 to the column unit 306 to wash the column.
  • the flow port 309a and the reaction tank 30 are communicated, and the pump 34 is operated to suck out the liquid (elution buffer (pH 8.0, 20 mmol / L Tris-HCl)) stored in the liquid storage unit 309 (step S1190).
  • the combined flow port 306a and the reaction tank 30 are communicated, and the pump 34 is operated to flow the elution buffer in the reaction tank 30 to the column unit 306. Then, the eluate does not flow out to the waste liquid tank 327 and diffuses. It accumulates on the path 327f (step S1200).
  • the squeezing of the tube by the pump 34 is stopped.
  • the amplified DNA adsorbed on the column is eluted into the elution buffer, a solution containing the amplified DNA is accumulated in the diffusion channel 327f.
  • the width of the diffusion channel 327f is substantially equal to the width of the column portion 306, and the degree of diffusion of DNA eluted in the elution buffer after flowing through the column is determined by this diffusion. This is larger than in the case of a channel having a narrower width than the width of the channel 327f. For this reason, the bias of the distribution of DNA in the elution buffer is reduced.
  • the reason why the width of the diffusion flow path 327f and the width of the column portion 306 are made substantially equal in this way is as follows.
  • step S1200 the pump 34 is operated to suck back the elution buffer from which DNA has accumulated in the diffusion channel 327f back into the reaction tank 30 (step S1210).
  • the width of the diffusion channel 327f is substantially equal to the width of the column portion 306, the degree of diffusion of the eluted DNA is large compared to the case where the width is narrow, and the elution buffer together with the DNA eluted in the reaction vessel 30 When the sucrose is sucked out, the eluted DNA hardly remains in the column portion 306.
  • the injection port 310a and the reaction tank 30 are communicated, and the pump 34 is operated to inject the elution buffer in the reaction tank 30 into the closed channel 310 (step S1220).
  • the air filled in the closed flow path 310 is compressed by the liquid to be injected and the pressure is increased.
  • the pressure applied to the reaction tank 30 by the pump 34 is adjusted, the volume of the closed channel 310 and the amount of the mixed solution injected into the closed channel 310 can be adjusted from the applied pressure. It is. For example, when the pressure is set to 202 kPa (2 atm), the mixed solution in an amount half the volume of the closed channel 310 can be injected.
  • step S1230 the flow port 309a and the reaction tank 30 are communicated, and the mixed solution remaining in the reaction tank 30 is discharged to the liquid storage unit 309 (step S1230).
  • the mixed solution is discharged into the liquid container 309.
  • the injection port 310a and the reaction tank 30 are communicated, and the mixed solution injected into the reaction channel 310 is supplied to the reaction tank 30 (step S1240) to obtain adjusted DNA.
  • the pressure in the reaction tank 30 is lowered, while the air in the closed channel 310 is injected with the mixed solution in step S1220. Keep the pressure when you are. For this reason, the mixed solution injected into the closed channel 310 is supplied to the reaction tank 30 due to the difference in pressure.
  • the pump 34 may be operated to supply the mixed solution into the reaction tank 30.
  • the CPU 42 of the controller 40 reads and executes the reaction processing routine stored in the flash ROM 43. This routine is executed after the above-described DNA adjustment processing routine is completed.
  • the CPU 42 first connects the flow port 311a and the reaction tank 30 having the adjusted DNA, operates the pump 34, and sucks out the liquid stored in the liquid storage unit 311 (step S1300). .
  • the cartridge 350 is rotated so that the closed port 312a and the reaction tank 30 are connected, and the temperature in the reaction tank 30 is maintained at 90 ° C.
  • step S1310 the temperature in the reaction vessel 30 is kept at 10 ° C. and stirred for 5 minutes (step S1320).
  • step S1320 the combined flow port 313a and the reaction tank 30 are communicated, and by adjusting the operation of the pump 34, the mixed solution stored in the reaction tank 30 is temporarily accumulated in the reaction flow path 365 of the miniarray 350b, and the Peltier element for cartridge
  • the reaction flow path 365 is kept at 42 ° C. for 60 minutes by 38a, and the probe DNA formed in the first and second spot areas 366a and 366b is allowed to undergo a hybridization reaction with the DNA in the mixed solution, and then the pump 34 is again used.
  • the depth of the reaction channel 365 is shallower as the width of the reaction channel 365 is wider, and becomes deeper as the width is narrower. Since the area is substantially constant and the first and second grooves 381a and 381b are provided, the mixed solution containing the target DNA due to the difference in location in the first and second spot areas 366a and 366b The difference in the flow rate of the probe DNA can be kept small, and the difference in the opportunity for the probe DNA to hybridize with the target DNA can also be kept small.
  • the mixed solution that has circulated through the miniarray 350b is accommodated in the waste liquid tank 328 through the above-described path.
  • the efficiency of the hybridization reaction can be improved by flowing the mixed solution after the mixed solution stored in the reaction tank 30 is accumulated in the reaction flow path 365 of the miniarray 350b. Thereby, reaction time can be shortened.
  • a flow path that gradually increases in width and gradually decreases in depth and a flow path that gradually increases in width and constant in depth are prepared, and the same probe DNA of the same concentration is provided in each flow path.
  • the flow port 315a and the reaction tank 30 are communicated, and the pump 34 is operated to suck out the liquid stored in the liquid storage unit 315 (step S1340).
  • the combined flow port 313a and the reaction tank 30 are communicated with each other, and the operation of the pump 34 is adjusted so that the cleaning liquid accommodated in the reaction tank 30 is temporarily stored in the reaction channel 365 of the miniarray 350b.
  • the interior is kept at 25 ° C. for 5 minutes by the Peltier element 38a for the cartridge, and the first and second spot areas 366a and 366b are washed.
  • the pump 34 is operated again to increase the pressure in the reaction tank 30 and the reaction flow path 365 is increased.
  • step S1350 the cleaning liquid once collected in step S1350 is discharged to the waste liquid tank 328. Subsequently, the same processing as in steps S1340 and S1350 is performed using the liquid stored in the liquid storage unit 316, and the first and second spot areas 366a and 366b of the mini array 350b are cleaned (steps S1360 to S1370). . Subsequently, the flow port 317a and the reaction tank 30 are communicated, and the pump 34 is operated to suck out the liquid stored in the liquid storage unit 317 (step S1380).
  • the combined flow port 313a and the reaction tank 30 are communicated, and the liquid stored in the reaction tank 30 is temporarily stored in the reaction flow path 365 of the miniarray 350b by adjusting the operation of the pump 34.
  • the pump 34 is operated again to increase the atmospheric pressure in the reaction tank 30, The liquid once stored is discharged to the waste liquid tank 328 (step S1390).
  • steps S1340 and S1350 is performed using the liquid stored in the liquid storage portions 318 and 319, respectively, and the first and second spot areas 366a and 366b of the mini-array 350b are cleaned (step S1400).
  • step S1430 the flow port 320a and the reaction tank 30 are communicated, and the pump 34 is operated to suck out the liquid stored in the liquid storage unit 320 (step S1440).
  • step S1440 the combined flow port 313a and the reaction tank 30 are communicated, and the liquid stored in the reaction tank 30 is temporarily stored in the reaction flow path 365 of the miniarray 350b by adjusting the operation of the pump 34. After the inside is kept at 25 ° C.
  • the DNA in the first and second spot areas 366a, 366b is subjected to a pigmentation reaction, and then the pump 34 is operated again to increase the atmospheric pressure in the reaction tank 30 and to the reaction flow path 365.
  • the liquid once stored is discharged to the waste liquid tank 328 (step S1450).
  • the flow port 321a and the reaction tank 30 are communicated, and the pump 34 is operated to suck out the liquid stored in the liquid storage unit 321 (step S1460).
  • the binding flow port 313a and the reaction vessel 30 are communicated, and the liquid contained in the reaction vessel 30 is caused to flow through the reaction flow path 365 of the miniarray 350b to allow the dye of the DNA in the first and second spot areas 366a and 366b.
  • the deposition reaction is stopped (step S1470).
  • DNA with pigments is obtained in the miniarray 350b (step S1480).
  • the lower member 363 of the mini array 350b is peeled off, and only the upper member 364 in which the first and second spot areas 366a and 366b are formed, for example, the mini array attachment 333 as shown in FIG.
  • the mini-array attachment 333 is set in an existing scanner and analyzed.
  • the thing for general DNA array analysis shall be used, and detailed description is abbreviate
  • the mini array attachment 333 is provided with a step portion 372 corresponding to the step portion 371 of the mini array 350b, and the step portions 371 and 372 are set so as to be matched. Further, the external shape of the mini-array attachment 333 is adapted to an existing scanner.
  • the miniarray attachment 333 in which the miniarray 350b is set is scanned with an existing scanner, for example, an OA scanner (GT-8700F, manufactured by Epson), and the rice deposition is detected from the pigmentation pattern that appears in the scanned image. Perform product type determination. It is also possible to visually determine the dye precipitation pattern. Since the mini-array 350b has two spot areas, the first and second spot areas 366a and 366b, when different probe DNAs are formed in the first and second spot areas 366a and 366b, More types of probe DNA can be formed than when only one of them is present. Therefore, the determination accuracy can be improved as compared with the case where only one of the first and second spot areas 366a and 366b is provided.
  • an existing scanner for example, an OA scanner (GT-8700F, manufactured by Epson)
  • GT-8700F manufactured by Epson
  • the mini-array 350b of this embodiment corresponds to the DNA array of the present invention
  • the first connection port corresponds to the inlet
  • the second connection port corresponds to the outlet
  • the reaction channel 365 corresponds to the reaction channel
  • the first The spot area 366a corresponds to the first probe formation region.
  • the second spot area 366b corresponds to the second probe formation region
  • the curved portion 365a corresponds to the curved portion
  • the straight portion 365b corresponds to the straight portion
  • the cartridge body 350a corresponds to the solution storage device
  • the flow path 328g corresponds to the supply port
  • the waste liquid tank 328 corresponds to the waste liquid tank
  • the flow path 328f corresponds to the discharge port.
  • the width of the reaction channel 365 is formed so as to gradually widen from the first connection port 361 toward the first spot area 366a, and the depth of the reaction channel 365 is increased.
  • the width of the reaction channel 365 is shallower and the depth of the reaction channel 365 is narrower. Therefore, the change in the cross-sectional area of the reaction channel 365 from the first connection port to the first spot area 366a is smaller than that in the case of having a constant depth regardless of the width, and the first spot area
  • the difference in the flow rate of the solution due to the difference in the location in 366a can be kept small.
  • the first spot area 366a after the hybridization reaction can be washed relatively evenly, and as a result, the whole can be washed with a small amount of washing solution.
  • reaction channel 365 has a substantially constant cross-sectional area from the first connection port 361 to the second connection port 362, the difference in the flow rate of the solution due to the difference in the location in the first spot area 366a is further increased. It can be kept small.
  • the reaction channel 36 has a U-shape in which the width of the curved portion 365a is narrower than the straight portion 365b.
  • the wider the U-shaped curved portion 365a the shallower the depth and the narrower the width. Since the depth is formed so deeply as compared with the case where the U-shaped curved portion has the same width and depth as the straight portion, the flow rate deviation of the solution at the U-shaped curved portion 365a is reduced. It can be kept small. For this reason, it is possible to suppress a difference in the chance of a hybridization reaction with the target DNA flowing through the reaction channel 365 between the plurality of types of probe DNAs in the second spot area 366b provided downstream of the curved portion 365a. .
  • the width of the reaction channel 365 is formed so as to gradually narrow from the second spot area 366b toward the second connection port 362, the solution that has passed through the second spot area 366 is allowed to react with the reaction flow. It is possible to discharge from the second connection port 362 in a state where the difference in flow velocity due to the difference in location in the path 365 is small.
  • the first groove 381a is provided on both sides of the first spot area 366a and the second groove 381b is provided on both sides of the second spot area 366b, when the solution passes through the first spot area 366a, Since the solution flows through the first spot area 366a along the solution flowing through the first groove 381a, it is possible to suppress a decrease in the flow velocity of the solution flowing through the end portion of the first spot area 366a.
  • the solution passes through the second spot area 366b
  • the solution flows through the second spot area 366b along the solution flowing through the second groove 381b. Therefore, the flow velocity of the solution flowing through the end portion of the second spot area 366b. Can be suppressed. For this reason, the difference in the flow rate of the solution due to the difference in the location in the first and second spot areas 366b can be further reduced.
  • the mini-array 350b is used by being mounted on the cartridge body 350a, a series of processes of the hybridization reaction between the probe DNA and the target DNA and the washing of the first and second spot areas 366a and 366b are performed as one. It can be executed relatively easily with the cartridge body 350a.
  • the mini array 350b includes the first and second spot areas 366a and 366b, but may include only one of them.
  • the reaction channel 365 includes the first and second grooves 381a and 381b. However, the reaction channel 365 without the first and second grooves 381a and 381b and having a rectangular cross section. It is good. At this time, the cross-sectional area between the first connection port 361 and the second connection port 362 may be substantially constant.
  • the reaction flow path 365 has a substantially constant cross-sectional area between the first connection port 361 and the second connection port 362 including the first and second grooves 381a and 381b. If the first and second grooves 381a and 381b are provided, the cross-sectional area need not be constant. Even in such a case, the difference in the flow rate of the solution due to the difference in the locations in the first and second spot areas 366a and 366b can be suppressed to be smaller than in the case where there is no groove. Therefore, it is possible to suppress a difference in the chance of performing a hybridization reaction with the target DNA flowing in the reaction channel between a plurality of types of probe DNAs formed in the width direction of the reaction channel.
  • the shape of the reaction channel 365 is U-shaped, but may be a shape other than the U-shape, such as a linear shape or an L-shape.
  • the mixed solution is temporarily accumulated in the reaction channel 365 and the reaction channel 365 is maintained at 42 ° C. for 60 minutes. While the path 365 is maintained at 42 ° C. for 60 minutes, the direction of the tube of the pump 34 may be continuously switched to vibrate the mixed solution in the reaction channel 365. In such a case, the mixed solution continuously flows in the first and second spot areas 366a and 366b during the hybridization reaction.
  • the effect of the present invention is further enhanced in that the difference in flow velocity due to the difference in location in the first and second spot areas 366a, 366b is suppressed to be small, and the difference in the chance of hybridization reaction due to the difference in location is reduced.
  • Step S1210 after the process of step S1210 in FIG. 14, an adjusted amount of the mixed solution is supplied into the reaction tank 30 using the closed channel 310 to obtain adjusted DNA.
  • Step S1220 to S1240 after the processing of Step S1210, the cartridge 350 is rotated so that the closing port 312a and the reaction tank 30 are connected, and the rotation direction and the number of steps of the stepping motor connected to the pump 34 (the number of rotations)
  • the pressure in the reaction tank 30 is set to 70 kPa, the temperature in the reaction tank 30 is maintained at 80 ° C. for 50 minutes, and the mixed solution in the reaction tank 30 is concentrated, It is good also as what obtains adjusted DNA.
  • the cartridge main body 350a is composed of four layers of the first layer 351a to the fourth layer 351d.
  • the cartridge main body 350a is formed of the four layers. It is not restricted to what becomes, It is good also as what consists of 3 layers, and it is good also as what consists of 5 layers.
  • the mini-array 350b is used to identify rice varieties, but may be used for other reactions. At this time, other reaction probe DNAs may be formed in the first and second spot areas 366a and 366b.
  • the cartridge body 350a may contain other reaction liquid.
  • the present invention can be used for gene sequence analysis, and can be used, for example, for diagnosis of diseases in the medical industry and diagnosis of varieties in the food industry.

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Abstract

La largeur d'un passage d'écoulement de réaction (365) d'une mini-puce (350b) est formée de façon à augmenter progressivement d'un premier orifice de connexion (361) vers une première zone de point (366a). selon l'invention, la profondeur du passage d'écoulement de réaction (365) est construite de telle sorte que plus la largeur du passage d'écoulement de réaction (365) est importante, plus la profondeur est faible, et plus la largeur du passage d'écoulement de réaction (365) est faible, plus la profondeur est importante. Ceci amène une variation de la zone en section transversale du passage d'écoulement de réaction (365) du premier orifice de connexion à la première zone de point (366a) à être inférieure à celle d'un boîtier dans lequel le passage d'écoulement de réaction (365) a une profondeur constante indépendamment de la dimension de la largeur du passage d'écoulement de réaction. Par conséquent, la différence entre les vitesses d'écoulement d'une solution à différentes positions dans la première zone de point (366a) peut être conservée inférieure à celle d'un boîtier dans lequel le passage d'écoulement de réaction a une profondeur constante indépendamment de la dimension de la largeur du passage d'écoulement de réaction.
PCT/JP2009/053576 2008-07-01 2009-02-26 Puce à adn WO2010001636A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002031638A (ja) * 2000-07-17 2002-01-31 Mitsubishi Chemicals Corp 生体試料検出用チップ及び生体試料検出方法
JP2002257830A (ja) * 2000-12-28 2002-09-11 F Hoffmann La Roche Ag カートリッジを振動させることにより核酸試料を処理する方法、並びにその方法を実施するための装置及びカートリッジ
JP2004130219A (ja) * 2002-10-10 2004-04-30 Kawamura Inst Of Chem Res マイクロ流体素子、流体処理デバイス、および流体処理方法
JP2005221478A (ja) * 2004-02-09 2005-08-18 Taisei Plas Co Ltd バイオチップの検出体とその製造方法
WO2006054689A1 (fr) * 2004-11-22 2006-05-26 Nissui Pharmaceutical Co., Ltd. Micropuce
JP2006226841A (ja) * 2005-02-17 2006-08-31 Fuji Photo Film Co Ltd 全反射減衰を利用したセンサユニット，測定方法及び装置
JP2008134188A (ja) * 2006-11-29 2008-06-12 Olympus Corp プローブ固相化反応アレイおよび該アレイの製造方法
JP2008134189A (ja) * 2006-11-29 2008-06-12 Olympus Corp プローブ固相化反応アレイおよび該アレイの製造方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002031638A (ja) * 2000-07-17 2002-01-31 Mitsubishi Chemicals Corp 生体試料検出用チップ及び生体試料検出方法
JP2002257830A (ja) * 2000-12-28 2002-09-11 F Hoffmann La Roche Ag カートリッジを振動させることにより核酸試料を処理する方法、並びにその方法を実施するための装置及びカートリッジ
JP2004130219A (ja) * 2002-10-10 2004-04-30 Kawamura Inst Of Chem Res マイクロ流体素子、流体処理デバイス、および流体処理方法
JP2005221478A (ja) * 2004-02-09 2005-08-18 Taisei Plas Co Ltd バイオチップの検出体とその製造方法
WO2006054689A1 (fr) * 2004-11-22 2006-05-26 Nissui Pharmaceutical Co., Ltd. Micropuce
JP2006226841A (ja) * 2005-02-17 2006-08-31 Fuji Photo Film Co Ltd 全反射減衰を利用したセンサユニット，測定方法及び装置
JP2008134188A (ja) * 2006-11-29 2008-06-12 Olympus Corp プローブ固相化反応アレイおよび該アレイの製造方法
JP2008134189A (ja) * 2006-11-29 2008-06-12 Olympus Corp プローブ固相化反応アレイおよび該アレイの製造方法

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