WO2002042430A1 - Extracteur à acide nucléique - Google Patents

Extracteur à acide nucléique Download PDF

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Publication number
WO2002042430A1
WO2002042430A1 PCT/JP2000/008338 JP0008338W WO0242430A1 WO 2002042430 A1 WO2002042430 A1 WO 2002042430A1 JP 0008338 W JP0008338 W JP 0008338W WO 0242430 A1 WO0242430 A1 WO 0242430A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
tank
disk
extraction device
centrifugal force
Prior art date
Application number
PCT/JP2000/008338
Other languages
English (en)
Japanese (ja)
Inventor
Naruo Watanabe
Yoshihiro Nagaoka
Yukiko Ikeda
Teruhisa Akashi
Yuji Miyahara
Original Assignee
Hitachi, Ltd.
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 Hitachi, Ltd. filed Critical Hitachi, Ltd.
Priority to PCT/JP2000/008338 priority Critical patent/WO2002042430A1/fr
Priority to JP2002545136A priority patent/JPWO2002042430A1/ja
Publication of WO2002042430A1 publication Critical patent/WO2002042430A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • 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
    • B01L3/502738—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 integrated valves
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/07—Centrifugal type cuvettes
    • 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
    • B01L2200/0621—Control of the sequence of chambers filled or emptied
    • 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
    • B01L2300/087—Multiple sequential chambers
    • 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
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • 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
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • 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
    • B01L3/502753—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 bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00178—Special arrangements of analysers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00465—Separating and mixing arrangements
    • G01N2035/00495—Centrifuges
    • G01N2035/00504—Centrifuges combined with carousels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0439—Rotary sample carriers, i.e. carousels
    • G01N2035/0446—Combinations of the above
    • G01N2035/0449—Combinations of the above using centrifugal transport of liquid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0439—Rotary sample carriers, i.e. carousels
    • G01N2035/0453—Multiple carousels working in parallel

Definitions

  • the present invention relates to a nucleic acid extraction device for extracting nucleic acids from cells, microorganisms, viruses, and the like in an aqueous solution.
  • nucleic acid extraction There is a genetic diagnosis that uses a nucleic acid to diagnose a subject from this pattern.
  • nucleic acids are extracted from body fluids, tissues, and the like collected from a subject, and diagnosed by pattern analysis of the nucleic acids. For this reason, nucleic acid extraction must be performed accurately.
  • Examples of conventional techniques relating to nucleic acid extraction include, for example, “Bio-experimental illustrations: Basics of molecular biology experiments (1995, Shujunsha) P113-P122”
  • the prior art 1 discloses a method in which an aqueous solution containing a nucleic acid is held in a small container called a tube, and this is manually injected with various treatment liquids, stirred, and repeatedly centrifuged. As a device that automates this operation, it is marketed as Toyobo Co., Ltd. (Nucleic acid automatic separation device, NS-100).
  • the method of holding and treating the aqueous solution containing nucleic acids and various solutions of the prior art 1 in a small container called a tube can increase the sample volume and perform simple processing.
  • Raw There is a problem that it is difficult to extract accurately.
  • a complicated mechanism such as a robot system is adopted to automate these operations, it is difficult to increase the equipment cost and improve the throughput of the processing.
  • the method of processing on a resin-made circular substrate of the prior art 2 is that the aqueous solution containing nucleic acids and various solutions are all held on one substrate, and high-precision extraction is possible without contamination by foreign substances.
  • An object of the present invention is to realize an accurate and highly sensitive nucleic acid extraction device that does not contaminate foreign substances in an apparatus for extracting nucleic acid from an aqueous solution containing nucleic acid, and to provide it at low cost. Disclosure of the invention
  • the present invention provides a rotating disk with an aqueous solution containing nucleic acid, such as blood, provided with a plurality of tanks separated by a flow path or a partition, and connecting the respective tanks by operating an opening / closing mechanism provided in the flow path or the partition. Then, by rotating the disk and operating the opening / closing mechanism, the nucleic acid was extracted while being sequentially moved for each component.
  • nucleic acid such as blood
  • all or a part of the flow path or the partition, the opening / closing mechanism, and the tank are made of a plastic material from which a part of the component is not eluted with respect to the chemical having a protein denaturing action.
  • a predetermined amount of a drug having a denaturing effect on the protein or a drug which is more easily bound to water molecules than nucleic acid is previously enclosed.
  • the drug having a protein denaturing action is a solution obtained by mixing guanidine thiosinate, buenol, phenol, black form and isoamyl alcohol in a ratio of 25: 24: 1, respectively.
  • it refers to chemicals that have the effect of breaking down a membrane made of a protein containing nucleic acids and extracting nucleic acids, such as a solution of chloroform and isoamyl alcohol mixed in a 24: 1 ratio.
  • a chemical that is easier to bind to a water molecule than a nucleic acid refers to a chemical such as ethanol or isopropyl alcohol that has an action of coagulating a nucleic acid dissolved in water when mixed with an aqueous solution containing a nucleic acid.
  • the aqueous solution containing nucleic acids is stored in the tank for each component, and therefore, the aqueous solution containing a large amount of nucleic acids is not limited to the volume per unit length of the flow path connecting them. Can be processed. 'In addition, since all processes can be automated, accurate extraction can be performed without contamination. In addition, the tank holding the aqueous solution is separated by a partition wall, and this tank is manufactured by plastic injection shaping, so that the cost can be reduced. And since it can be used up in one extraction, there is no measurement error due to the difference of the measurement target.
  • FIG. 1 shows that nucleic acid is dissolved from an aqueous solution containing nucleic acid according to the first embodiment of the present invention.
  • FIG. 2 is an external view of a device for extracting nucleic acids from an aqueous solution containing nucleic acids according to the first embodiment
  • FIG. 2 is an external view for explaining a mechanism for centrifuging or stirring the device
  • FIG. FIG. 4 is a view showing a procedure for extracting a nucleic acid from an aqueous solution containing a nucleic acid using the device according to the first embodiment
  • FIG. 4 is a diagram showing a device for extracting a nucleic acid from an aqueous solution containing a nucleic acid according to the second embodiment.
  • FIG. 5 is a diagram showing the relationship between the operation of a mechanism for centrifuging or agitating the device and the operation of an opening / closing mechanism in Fig. 5;
  • FIG. 6 is a diagram illustrating the function of a device for extracting nucleic acid from an aqueous solution containing nucleic acids according to the first embodiment.
  • FIG. 7 is a diagram illustrating the function of a device containing nucleic acids according to a second embodiment of the present invention. Debye to extract nucleic acid
  • FIG. 8 is a diagram showing a procedure for extracting a nucleic acid using a device for extracting a nucleic acid from an aqueous solution containing a nucleic acid according to the second embodiment, and FIG.
  • FIG. 9 is a diagram showing a procedure for extracting the nucleic acid according to the second embodiment.
  • FIG. 4 is a diagram showing the relationship between the operation of a mechanism for centrifuging or agitating the device and the operation of an opening / closing mechanism in a device for extracting nucleic acid from a contained aqueous solution.
  • FIG. 1 (a) shows a plurality of tanks formed on a rotating disk and connected by a flow path having an opening / closing mechanism for extracting nucleic acids from an aqueous solution containing nucleic acids.
  • human whole blood is particularly described as an example of an aqueous solution containing nucleic acids.
  • biological fluids containing nucleic acids or aqueous solutions in which tissues containing nucleic acids are suspended in water. Can be extracted.
  • FIG. 1 (b) is an enlarged view of a portion of the tank and the flow channel in FIG. 1 (a).
  • the disk 100 has a plurality of tanks formed therein.
  • the disk 100 is provided with a mounting hole 102 for being mounted on a drive mechanism and rotated.
  • Disk 1 0 0 has the following layers.
  • a sample holding tank 104 for holding whole blood as a sample for analysis, a separation tank 106 for separating serum from whole blood, and an extraction for removing nucleic acids from a nuclear wall containing nucleic acids
  • the sample holding tank 104 and the separation tank 106 are connected by a flow path 114.
  • the separation tank 106 and the extraction tank 108 are connected by a flow path 116.
  • the flow path 1 16 is provided with an opening / closing mechanism 122 for controlling the movement of the solution on the way.
  • the flow path 118 connects the extraction tank 108 and the recovery tank 110, and includes an opening / closing mechanism 124 for controlling the movement of the solution in the middle thereof.
  • the flow path 120 connects the collection tank 110 and the nucleic acid holding tank 112 and has an opening / closing mechanism 126 for controlling the movement of the solution in the middle thereof.
  • Wiring 1 2 8, wiring 1 3 0, and wiring 1 3 2 are connected to the disc 1 0 0 through the hole 10 2 to control the respective opening and closing mechanisms 1 2 2, 1 2 4 and 1 2 6. Connected to an external (not shown) controller.
  • the opening / closing mechanism of this embodiment is configured by an electromagnetic valve that opens and closes with an electromagnetic force
  • the present invention is not limited to this method, and a system that switches gears and the like with a single drive system to open and close may be used.
  • the sample holding tank 104 is provided with a hole 103 for injecting whole blood.
  • Each tank except for this hole and each flow path are sealed to shut off outside air, preventing foreign matter from entering.
  • Fig. 2 shows a mechanism for generating centrifugal force by rotating the disk 10 ° including the tank and a mechanism for stirring the solution contained in the tank in the disk 100 °. .
  • the disk 100, the rotating shaft 200 and the motor 202 are mounted on the disk 204.
  • the disk 204 is rotationally driven by a rotating shaft 206 of the motor 208.
  • the center of the rotation axis 200 of the disk 100 and the center of the rotation axis 206 of the disk 204 are attached with a predetermined distance therebetween. In this way, it is necessary to provide a rotating shaft at intervals.
  • the centrifugal force applied to each tank can be increased. That is, while rotating around the rotating shaft holding the disk 100, the whole of them is driven in a so-called planetary motion in which the center of the rotating shaft is shifted from the rotating shaft 206 of the disk 204.
  • the disk 204 is provided with wires 2 1 2, 2 1 4 and 2 16, which are connected to the wires 1 2 8 1 3 0 and 1 3 2 provided on the rotating disk 100. ing. In addition, wirings 218 and 220 for driving and controlling the motor 202 are provided on the disk 204. The operation of each opening / closing mechanism on the disk 100, the motor, and the motor 208 for rotating the disk 204 is centrally controlled by the control device 230 via the control line 232. .
  • FIGS. 3 (a) to 3 (f) are diagrams for explaining the operation of extracting nucleic acids from whole blood.
  • FIGS. 3 (a) to 3 (f) there is a protein denaturing effect for removing nucleic acids from the nuclear wall encapsulating nucleic acids, which is not shown in FIG. 1 or FIG.
  • a predetermined amount of the drug 300 is held in the extraction tank 108, and the drug 302, which is easier to bind to water molecules than the nucleic acid, is stored in the recovery tank 110.
  • the arrow 100 0 indicates the direction of the centrifugal force generated by the rotation of the disk 100.
  • FIG. 3 (a) shows a state in which the disk 100 is stationary. At this time, 310 whole blood collected is injected into the sample holding tank 104 from the injection hole 103.
  • FIG. 3 (b) shows the case where the disk 100 is rotated near the separation tank 106 so that the centrifugal force becomes 150 G or more.
  • the whole blood 310 moves from the sample holding tank 104 to the separation tank 106.
  • substances 3 14 other than serum are distributed on the outer periphery of the disc 100 and serum 3 12 is distributed on the rotation center side in the separation tank 106 by centrifugal force.
  • the higher the rotation speed of the disk 100 the more the separation tank 106 The time for separating serum and components other than serum can be shortened.
  • the outlet of the flow path 114 in the separation tank should be It is desirable that the entrance of the flow path 1 16 be located on the side of the center of rotation of the disk 100.
  • the separated serum 312 passes through the flow path 1 16, passes through the opening / closing mechanism 1 2 2 previously opened, and moves to the extraction tank 108.
  • FIG. 3 (c) shows a state in which nucleic acids are extracted from the serum 312 in the extraction tank 108.
  • a predetermined amount of a drug 300 having a protein denaturing action is held in the extraction tank 108 in advance, and the drug 300 reacts with the serum 112.
  • phenol is used as the drug 300 having a protein denaturing effect
  • serum is sent, phenol 300 is initially converted to an oil layer in the extraction tank 108, and serum 12 is in a separated state as an aqueous layer.
  • the amount of phenol 300 is preferably equal to or greater than the amount of serum 112 to be reacted.
  • disk 204 is also in liquid. It was decided to rotate and stir at 08. At this time, the respective opening / closing mechanisms 122 and 124 in the middle of the flow path 116 and the flow path 118 are closed. The centrifugal force generated by the rotation of both disks is synthesized in the extraction tank 108 on the disk 100. For this reason, the rotation speed of the disk 100 and the disk 204 needs to be a value such that the direction of the centrifugal force does not depend on the rotation angle of the disk 100.
  • the extraction tank 108 is provided at a position 45 mm away from the center of the rotation axis 200 (the diameter of the extraction tank 108 is 6 mm), and the rotation axis 200 is the rotation axis. It is located at a distance of 51 mm from 206.
  • the rotation speed of the disk 100 is set to 500 rpm
  • the rotation speed of the disk 204 may be set to be higher than 200 rpm rp'm.
  • the shape of the extraction tank 108 is shown in Fig. 1. In addition to the circular shape shown in FIG. 3, if the shape is a polygon such as a square, the effect of extracting nucleic acids is enhanced.
  • FIG. 3 (d) shows that after the operation of extracting nucleic acids from serum 312 shown in FIG. 3 (c), the rotation of the disk 204 shown in FIG.
  • This figure shows the state when 100 is rotated near the extraction tank 1108 so that the centrifugal force becomes 150 G or more.
  • the phenol 300 has a small centrifugal force in the portion where the centrifugal force increases, and the aqueous solution 3 In between, albumin and globulin, which are proteins in serum (not shown) denatured by phenol 300, are distributed in layers.
  • FIG. 3 (e) shows a so-called centrifugal separation operation of taking out a very small amount of the solution having an increased nucleic acid concentration from the aqueous solution 316 containing the nucleic acid.
  • the recovery tank 110 previously holds a drug 302 that is easier to bind to water molecules than nucleic acids. Then, the chemical 300 and the aqueous solution 316 containing nucleic acid are reacted.
  • ethanol alcohol when ethanol alcohol is used as a drug 302 that is easier to bind to water molecules than nucleic acids, water molecules in the aqueous solution 3 16 and molecules of ethanol alcohol 302 bind to dissolve the nucleic acids. The amount of water that was being used is reduced. For this reason, nucleic acids are precipitated. Furthermore, since the density of the nucleic acid is higher than that of the surrounding water or ethanol alcohol 302, the centrifugal force 1 (precipitates in the direction of 300. The larger the centrifugal force 100, the faster the nucleic acid becomes. Is desirably 1000 g or more. The amount of ethanol alcohol 302 is 2.5 times larger than that of aqueous solution 3 16 to be reacted with ethanol.
  • the recovery tank 110 is designed so that its volume is larger than that of the extraction tank 108 and the separation tank 106. It is a deeper tank in size.
  • this amount may be equal to the amount of the aqueous solution 316 to be reacted therewith.
  • the location where the centrifugal force 1000 becomes maximum is near the opening / closing mechanism 126 in the middle of the flow path 120.
  • FIG. 3 (f) shows the operation of moving the nucleic acid precipitated near the opening / closing mechanism 126 in the middle of the channel 120 to the nucleic acid holding tank 112 together with the nearby aqueous solution. It is. Also at this time, it is desirable that centrifugal force of 1000 G or more is applied to the recovery tank 110. By opening and closing the opening and closing mechanism 126 in the middle of the flow path 120 for a very short time under this gravity, the solution having a high nucleic acid concentration can be moved to the nucleic acid holding tank 112.
  • FIG. 4 (a) shows the control sequence of each motor and the open / close mechanism.
  • the control device 230 drives the disk 210 to rotate the motor 210, the motor 204 rotates the disk 204, and the opening / closing mechanism 122 on the disk 100.
  • 4 and 1 26 show the operation status in the processing state.
  • the positional relationship of each tank on the disk 100 is as shown in Fig. 4 (b).
  • the distance between the center of the rotation axis 200 and the center of the rotation axis 206 is 51 mm.
  • the horizontal axis of the graph is not real time, but the processing content shown at the bottom of the graph.
  • the amount of nucleic acid contained in the solution in the nucleic acid holding tank 112 is proportional to the amount of whole blood injected into the sample holding tank 104. And increase.
  • the amount of the solution in the nucleic acid holding tank 112 is limited to the amount of the solution localized near the opening / closing mechanism 126 in the middle of the channel 120. Therefore, when the amount of nucleic acid contained in the collected whole blood 310 is small, the amount of nucleic acid contained in the finally obtained solution 320 can be increased by increasing the amount of whole blood. Highly sensitive nucleic acid extraction can be performed.
  • the amount of nucleic acid contained in whole blood 310 when the amount of nucleic acid contained in whole blood 310 is large, the amount of nucleic acid contained in final solution 320 can be adjusted by reducing the amount of whole blood. It can handle whole blood with a wide range of nucleic acid concentrations. In addition, since the whole blood, from 310 to solution containing a large amount of nucleic acid, is processed in a plurality of tanks and a flow path connecting them, accurate extraction is possible without the risk of foreign matter entering. it can.
  • each tank, each flow path, and the inlet port 103 set on the circle 100 can be integrally formed by directly cutting out the material forming the disc 100. it can. If they are integrally formed, the disk 100 can be made smaller and the processing cost can be reduced. This means that the disc 100 can be easily made disposable, and the effect of eliminating contamination between samples can be obtained.
  • Polypropylene or fluororesin is suitable as a material for forming the disk 100.
  • any material that does not dissolve its components in whole blood and is not violated by a drug 300 having a protein denaturing effect can be applied.
  • a polycarbonate resin having good formability is used as a material for forming the tank and the flow path, if this surface is coated with a fluororesin, the strength of the disk 100 and the intrusion of foreign matter during processing can be avoided. And the disk 100 can be formed at low cost.
  • a material for forming the tank / flow path a material that elutes trivalent ions such as aluminum can be used. By using the material, a three-dimensional shape such as a tank can be pressed. Production costs can be reduced.
  • each tank and each flow path provided on the disk 100 are formed separately by injection molding of polypropylene, and these are arranged on the disk 100 and connected by bonding. May be manufactured. When manufactured in this manner, the volume of each tank can be adjusted or selected according to the sample, the processing conditions in each tank can be optimized, and the nucleic acid can be extracted efficiently. can get.
  • FIG. 5 shows another embodiment.
  • a new flow path 1 16 0 and an opening / closing mechanism 1 2 are provided between the extraction tank 108 of the previous embodiment (referred to as the first extraction tank in this embodiment) and the collection tank 110.
  • 20 and a second extraction tank 1 080 were installed.
  • phenol is previously stored as a drug 30000 having a protein denaturing action.
  • the aqueous solution containing the nucleic acid extracted in the first extraction tank 1108 is re-extracted with a chemical 30000.
  • FIG. 6 shows the configuration of still another embodiment.
  • the collection tank 110 (referred to as the first collection tank in the present embodiment) and the nucleic acid holding tank 112, a new flow path 1200 and an opening / closing mechanism 124 are added.
  • This is a configuration in which two tanks 110 are installed.
  • ethanol alcohol 302 is held in advance similarly to the first collection tank 110, and the water containing nucleic acid is stored in the first collection tank 110.
  • an injection hole 103 for injecting whole blood as a sample and one processing equipment from the sample holding tank 104 to the nucleic acid holding tank 112 are provided on the disk 100.
  • the same effect can be obtained by installing a plurality of the devices.
  • Providing a plurality of processing facilities on a single disk has the effect of increasing the number of processes per unit time in the process of extracting nucleic acids from aqueous solutions containing a plurality of nucleic acids.
  • FIG. 7 shows a configuration in which a plurality of rooms are provided on a disk in a circumferential direction to extract nucleic acids from an aqueous solution containing nucleic acids, and a partition between the rooms is provided with an opening / closing mechanism.
  • the disk is described as rotating clockwise.
  • the disk 700 is provided with a plurality of tanks (rooms) partitioned by partition walls in the rotation direction and the circumferential direction.
  • the disk 700 is provided with a hole 720 for mounting on a mechanism (rotary shaft) for rotating the disk 700.
  • the disc also has a sample holding tank 706 for holding whole blood, a separation tank 712 for separating serum from whole blood, and a nucleic acid for removing nucleic acids from a nuclear wall containing nucleic acids.
  • a nucleic acid holding tank 736 for holding the prepared nucleic acid.
  • the separation tank 712 provided on the outer peripheral side of the sample holding tank 706 is fractionated by a partition wall 707, and both are controlled by a centrifugal force as an opening / closing mechanism and a spring 710.
  • a sealer 708 is connected by Separation tank 7 12 and extraction tank 7 18 .
  • Separation tank 7 12 and extraction tank 7 18 are separated by partition wall 7 17, and both are connected by a centrifugal force as an opening and closing mechanism and a seal 7 14 controlled by a spring 7 16 .
  • Extraction tank 7 1 8 and The recovery tank 724 is fractionated by a partition wall 723, and is connected by a sealer 702 and a sealer 721 controlled by a centrifugal force as an opening / closing mechanism and a spring 722. .
  • partition wall 723 is also used as a partition wall of the sample holding tank 706 and the recovery tank.
  • the collection tank 724 and the pharmaceutical tank 730 are separated by a partition 729, and both are connected by a centrifugal force as an opening / closing mechanism and a sealer 726 controlled by a panel 728.
  • the recovery tank 724 and the nucleic acid holding tank 736 are connected by a seal 732 controlled by a centrifugal force as an opening / closing mechanism and a spring 734.
  • An injection hole 704 for injecting whole blood is provided on the upper surface of the sample holding tank 706.
  • FIG. 8 (a) With the disk 700 still, the whole blood 750 is injected through the injection hole 704. At this time, the sealer 708 is closed, and the whole blood 750 is confined in the sample holding tank 706. In addition, in the extraction tank 718, a drug 755 having a protein denaturing action is held in advance. In the chemical tank 730, a drug 754 which is easier to bind to water molecules than nucleic acid is held. Next, the disk 700 is rotated (clockwise rotation). Then, as shown in FIG.
  • the serum 758 overflows from the open end of the sealer 714 to the extraction tank 718. To go. 'this At this time, if the opening interval of the sealer 714 is set so that components 756 other than red blood cells cannot exceed the sealer 714, only the serum moves to the extraction tank 718. become.
  • the serum 758 has been moved to the extraction tank 718, and the rotation speed of the disk 700 has been reduced. It shows a closed state.
  • a predetermined amount of a drug 752 having a protein denaturing action is held in the extraction tank 718 in advance.
  • the phenol 752 is separated as an oil layer and the serum 758 is separated as an aqueous layer.
  • the amount of phenol 752 is desirably equal to or greater than the amount of serum 758 to be reacted therewith.
  • phenol 752 and serum 758 are stirred to extract nucleic acids in serum 758. Therefore, the disk 100 in Fig. 2 is replaced by the disk 700, and in addition to the state in which the disk 700 is rotated by the motor 202 around the rotation axis 200, these are mounted.
  • the disc 204 thus formed was rotated by a motor 208 around a rotation axis 206 to stir using planetary motion.
  • the rotation speed of the disk 700 and the disk 204 is the centrifugal force generated by the rotation of the disk 700 and the disk 204 at the outermost periphery of the disk 700 of the extraction tank 718.
  • a value is used so that the direction of the synthesized centrifugal force does not depend on the rotation angle of the disk 700.
  • the rotation speed of the disk 700 is 500 rpm
  • the rotation speed of the disk 204 may be higher than 2000 rpm. However, the rotation speed is set so that the sealers 708 and 714 are closed.
  • FIG. 8 (d) shows an operation of transferring the aqueous solution 760 containing a large amount of nucleic acid distributed in the extraction tank 718 to the recovery tank 724.
  • the disk 700 has a greater number of rotations than the phenol 752 which is a chemical having a protein attribute action in the extraction tank 718 and the aqueous solution 760 containing nucleic acid.
  • the rotation speed is higher than the number of rotations for separating serum 758 and components 756 other than red blood cells, and a centrifugal force of about 300 G is generated in the collection tank.
  • the sealer 7 2 1 moves according to the centrifugal force, A gap is generated between the sealing element 720 and the aqueous solution 760 from the gap to the recovery tank 724.
  • a gap is also formed between the sealer 708 and the sealers 7 and 14 at the same time, but components 756 other than erythrocytes and chemicals 752 that have a protein denaturing action It does not move from 18.
  • FIG. 8 (e) shows an operation of taking out a very small amount of a solution having a high nucleic acid concentration from an aqueous solution 60 containing nucleic acids held in the recovery tank 724.
  • the rotation speed of the disk 700 is set to be higher than the rotation speed of the disk 700 in FIG. 8 (d) so that a centrifugal force of approximately 400 G is generated.
  • the sealer 720 and the sealer 721 connecting the extraction tank 718 and the recovery tank 724 the sealer 721 falls down according to the centrifugal force.
  • the sealer 720 is lowered so as to close the gap with the sealer 721, and the recovery tank 724 is sealed while holding the aqueous solution 760.
  • the centrifugal force be 1000 G or more.
  • the amount of ethanol alcohol 754 is desirably 2.5 times or more of the aqueous solution 760 to be reacted therewith.
  • the recovery tank 724 is deeper in size than the extraction tank 718 and the separation tank 712 so as to have a larger volume.
  • Fig. 8 (f) shows that the nucleic acid precipitated by centrifugal force in the aqueous solution 762 held in the recovery tank 724 and the slight aqueous solution around it are transferred to the nucleic acid holding tank 736.
  • FIG. With a centrifugal force of 1000 G or more, the sealer 732 lowers, and the nucleic acid precipitates and the solution having a higher concentration moves to the nucleic acid holding tank 736. If the centrifugal force is 1000 G or more, the sealer 732 remains lowered, and the nucleic acid continues to accumulate in the nucleic acid holding tank 7336 during centrifugation with this centrifugal force.
  • Fig. 9 (a) shows the motor 210 that the control device 230 rotates the disk 700, the motor 208 that rotates the disk 204, and the control device 230 on the disk 700. It shows the operation of the seals 7 08, 7 14, 7 20, 7 21, 7 26, and 7 36. However, the positional relationship of the sealers 7 08, 7 14, 7 20, 7 21, 7 26, and 7 36 on the disk 700 is as shown in Fig. 9 (b). It is.
  • the distance between the rotation axis 200 and the rotation axis 206 is 53 mm
  • the diameter of the disk 700 is 100 mm.
  • the horizontal axis of the graph is not real time, but the processing contents shown at the bottom of the graph.
  • the tank formed on the disk 700 is partitioned by the partition walls, the amount of whole blood to be processed can be further increased, and the highly sensitive nucleic acid can be obtained. An extraction can be performed. Also, if the entire disc 700 including each tank is manufactured by injection molding of polypropylene or the like, the production cost of the disc 700 can be reduced. Further, if the entire disc 700 including each tank is extruded and shaped with polycarbonate resin, and the surface is coated with a fluororesin, the strength of the disc 700 and entry of foreign matter during processing can be avoided. Furthermore, the disk 700 can be constructed at low cost. This is effective even when the amount of whole blood to be processed is small.If the polycarbonate resin surface is embossed to form each tank and the surface is coated with fluororesin, a disk 700 can be constructed at lower cost. can do.
  • the same effect can be obtained even if a plurality of processing facilities from the hole 704 and the sample holding tank 706 to the nucleic acid holding tank 736 are installed on the disk 700. Wear. By providing a plurality of processing facilities, the effect of increasing the number of processes per unit time in the process of extracting nucleic acids from an aqueous solution containing a plurality of nucleic acids can be obtained.

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Abstract

La présente invention concerne un extracteur à acide nucléique construit pour l'extraction d'acide nucléique par un déplacement d'une solution contenant de l'acide nucléique. A cet effet, on dispose d'une pluralité de bacs sur un disque rotatif, on relie les bacs entre eux au moyen d'un chemin d'écoulement équipé d'un mécanisme d'ouverture et fermeture, puis on sépare et on extrait séquentiellement des compositions spécifiées dans les solutions des bacs respectifs. L'invention concerne également un traitement automatique mettant en oeuvre une pluralité de bacs reliés par des chemins d'écoulement pourvus de mécanisme d'ouverture et fermeture. Ce procédé permet de traiter une solution aqueuse contenant de grandes quantités d'acides nucléiques sans qu'il y soit mélangé de corps étranger, de façon à garantir une extraction ultrasensible et très précise de l'acide nucléique et à réduire le prix et la durée de l'extraction de l'acide nucléique.
PCT/JP2000/008338 2000-11-27 2000-11-27 Extracteur à acide nucléique WO2002042430A1 (fr)

Priority Applications (2)

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PCT/JP2000/008338 WO2002042430A1 (fr) 2000-11-27 2000-11-27 Extracteur à acide nucléique
JP2002545136A JPWO2002042430A1 (ja) 2000-11-27 2000-11-27 核酸抽出装置

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PCT/JP2000/008338 WO2002042430A1 (fr) 2000-11-27 2000-11-27 Extracteur à acide nucléique

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004212050A (ja) * 2002-05-08 2004-07-29 Hitachi High-Technologies Corp 化学分析装置及び遺伝子診断装置
US20110244522A1 (en) * 2010-03-30 2011-10-06 Tae Seok Seo Rotational pcr equipment and pcr method using the same

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0084983A1 (fr) * 1982-01-14 1983-08-03 Jean Guigan Dispositif de conditionnement pour analyses multiples
EP0106536A2 (fr) * 1982-09-15 1984-04-25 Ortho Diagnostic Systems Inc. Dispositif centrifuge de séparation, de lavage et d'examen de microparticules et méthode d'utilisation
EP0187699A2 (fr) * 1985-01-07 1986-07-16 Btg International Limited Appareil automatique de chimie
JPH0335144A (ja) * 1989-06-30 1991-02-15 Nippon Shoji Kk ロータリー・キューベット
EP0569115A2 (fr) * 1992-05-05 1993-11-10 General Atomics Système d'élaboration d'ADN à grand débit

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0084983A1 (fr) * 1982-01-14 1983-08-03 Jean Guigan Dispositif de conditionnement pour analyses multiples
EP0106536A2 (fr) * 1982-09-15 1984-04-25 Ortho Diagnostic Systems Inc. Dispositif centrifuge de séparation, de lavage et d'examen de microparticules et méthode d'utilisation
EP0187699A2 (fr) * 1985-01-07 1986-07-16 Btg International Limited Appareil automatique de chimie
JPH0335144A (ja) * 1989-06-30 1991-02-15 Nippon Shoji Kk ロータリー・キューベット
EP0569115A2 (fr) * 1992-05-05 1993-11-10 General Atomics Système d'élaboration d'ADN à grand débit

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GREGORY J. KELLOGG ET AL.: "Centrifugal microfluidics: Applications", MICRO TOTAL ANALYSIS SYSTEM 2000 PROCEEDINGS, May 2000 (2000-05-01), pages 239 - 242, XP002936553 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004212050A (ja) * 2002-05-08 2004-07-29 Hitachi High-Technologies Corp 化学分析装置及び遺伝子診断装置
US20110244522A1 (en) * 2010-03-30 2011-10-06 Tae Seok Seo Rotational pcr equipment and pcr method using the same
US8906624B2 (en) * 2010-03-30 2014-12-09 Korea Advanced Institute Of Science And Technology (Kaist) Rotational PCR equipment and PCR method using the same

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