WO1998043724A1 - Appareil d'isolement - Google Patents

Appareil d'isolement Download PDF

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Publication number
WO1998043724A1
WO1998043724A1 PCT/JP1998/001455 JP9801455W WO9843724A1 WO 1998043724 A1 WO1998043724 A1 WO 1998043724A1 JP 9801455 W JP9801455 W JP 9801455W WO 9843724 A1 WO9843724 A1 WO 9843724A1
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WIPO (PCT)
Prior art keywords
nucleic acid
trap
separation
trapping
forced dispersion
Prior art date
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PCT/JP1998/001455
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English (en)
Japanese (ja)
Inventor
Jun Tomono
Junichi Mineno
Ikunoshin Kato
Original Assignee
Takara Shuzo Co., Ltd.
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Filing date
Publication date
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Publication of WO1998043724A1 publication Critical patent/WO1998043724A1/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
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/147—Microfiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/16—Feed pretreatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/18—Apparatus therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/04—Specific process operations in the feed stream; Feed pretreatment

Definitions

  • the present invention relates to a separation apparatus, a method for separating biological substances such as nucleic acids and proteins, and the like.
  • Nucleic acids can be broadly classified into RNA and DNA, and can be further classified into several types according to their functions.
  • plasmid is a parasitic replicon that grows in cells independently of its host chromosome, and is widely used as a vector (gene carrier) in recombinant DNA experiments.
  • a common purification method is the alkaline lysis method of Baumboim H.C. and Dolly J. [Birmboim, HC, Doly, J., Nucleic Acids Research, Vol. 7, No. 1513- 1523 (1979)] and the boiling method using Holms D. S. Hoka CHolmes. DS et al., Analytical Biochemistry, 114, 193 (1981)], etc. It is.
  • genomic DNA which encodes the natural genetic information of each organism
  • various methods have been developed as methods for purifying genomic DNA.
  • extraction and purification of DNA from nucleated cells such as animals can be carried out by the methods of Mamer N. and Sugar Ford D.W. Marmur, N., Stafford, DW, Nucleic Acids Research, Vol. 3, pp. 2303 (1975), and its modifications [1 1982, Cold Spring Herba La Lab. , T. Maniat is, et al., Molecular 'Cloning, A Laboratory' Manual (Molecular Cloning, A Laboratory) Manual), pp. 86-96].
  • the above method requires the use of hazardous solvents such as phenol and black-mouthed form to remove other components mixed in the cells, for example, proteins.
  • Various methods have been developed. For example, after lysing cells with an appropriate detergent, treating the lysate with an appropriate nuclease and protease, and further adding a chaotropic substance to denature and remove residual proteins from nucleic acids, then dialyzing the solution A method for preparing a nucleic acid by the method [JP-A-63-91093].
  • a buffer in which a chaotropic substance is dissolved is used as a buffer.
  • a method for separating and purifying nucleic acids by using a solvent-insoluble carrier that selectively adsorbs nucleic acids in a buffer solution is exemplified.
  • the method for example, an E. coli-containing solution obtained by subjecting a colon bacterium having a plasmid to ALD-SDS treatment by the method of Bamboim et al. L i C 1) The method of Marko et al.
  • nucleic acid adsorbent refers to a solvent-insoluble carrier that selectively adsorbs nucleic acids in the presence of a chaotropic substance, unless otherwise specified.
  • chaotropic anions dissolve agarose gel
  • a method for purifying nucleic acids present in agarose gel using a chaotropic substance and glass particles or glass fiber [Vogelstein, B. (Vogelstein, B. , Et al., Proceedings of the National Academy of Sciences of the United States of America, Vol. 76, pp. 61-56, Procedings of the National Academy of Sciences of the United States of America. 19 (1 979), Carton WC, et al., Analytical Biochemistry, Vol. 101, pp. 339-341 (1980)] have also been reported.
  • An object of the present invention is to improve a trap device used in a conventional method for separating a biological material, and to solve the above-mentioned problems. Disclosure of the invention
  • the first invention of the present invention is a method for torping an object to be trapped in a mixture.
  • the second invention of the present invention relates to a biological material using the separation device of the first invention. It concerns the separation method.
  • the third invention of the present invention comprises: (a) a step of lysing cells and forcibly dispersing a cell lysate to obtain a crude nucleic acid solution;
  • step (c) a step of eluting the nucleic acid from the nucleic acid adsorbent having the nucleic acid trapped in the step (b) under forced dispersion
  • a fourth invention of the present invention relates to a kit for separating and purifying a nucleic acid, which comprises at least a nucleic acid adsorbent and is used in the method for separating and purifying a nucleic acid according to the third invention of the present invention.
  • FIG. 1 is a schematic diagram showing a structure of a trap container for a cell lysis step.
  • 1 indicates a trap filter for the cell lysis step
  • 2 indicates a container.
  • FIG. 2 is a schematic diagram showing the structure of a trap container for a nucleic acid separation step.
  • 3 indicates a trap filter for a nucleic acid separation step
  • 4 indicates a container.
  • FIG. 3 is a schematic diagram showing the structure of one example of the separation device of the present invention.
  • reference numeral 5 denotes a trap container
  • 6 denotes a trap container fixing device
  • 7 denotes a rotary shaking device
  • 8 denotes a pressure reduction generator
  • 9 denotes a waste liquid receiving tray.
  • a trap device for trapping a trap target in a mixture and a forced dispersion force is applied to a trap target and a liquid containing Z or a trap target before trapping by the trap device.
  • a forced dispersion device for dispersing the trapping object and the Z or trapping object before the trapping and a separation device for separating the target object from the mixture.
  • Examples of the mixture include, but are not limited to, animal cells, plant cells, mucosa cells, cultures of living tissues and the like, lysates thereof, and chemically synthesized products.
  • examples of the lysate include those in which animal cells, plant cells, bacterial cells, biological tissues, and the like are disrupted by a conventional method and dissolved in an appropriate buffer.
  • the term “trap target” refers to a substance that is trapped in each step when a target substance is separated from a mixture.
  • separating nucleic acids it refers to “cells” trapped from a cell culture, “nucleic acids” trapped from a cell lysate, and the like.
  • the “target substance” is a substance intended to be separated from a mixture, such as a nucleic acid, refers to proteins, peptides, antibodies, compounds and the like.
  • the trap device of the separation device may be any device that can selectively trap only the trap target from the mixture. For example, to selectively trap cells from a cell culture solution, besides a device for trapping cells by applying a centrifugal force to a vessel containing the cell culture solution to precipitate the cells and removing the supernatant, Hole diameter
  • the cell culture solution is placed in a container holding a trap filter, such as a porous silica glass membrane fiber having pores of 0.3 to 1.0 ⁇ m, and the cells are filtered by means of vacuum filtration, pressure filtration or centrifugal filtration.
  • a device for trapping on the top is exemplified.
  • the trap container used in the device may be a cylindrical container holding a trap filter, and the properties and shape of the trap container may be determined by ordinary separation methods such as vacuum filtration, pressure filtration, and centrifugal filtration.
  • the filter and the container for holding the filter and the trap filter may be of an assembling type or an integral type as long as they can be used and have a shape.
  • An apparatus using a trap filter is preferable in terms of reducing the size and weight of the apparatus.
  • a trap device using vacuum filtration is preferable in terms of simplifying the structure.
  • the forced dispersion device of the separation device according to the present invention is, for example, used for dispersing a trapped object trapped by the trap device in a liquid.
  • a device for dispersing examples include a shaking device, an ultrasonic generator, and a publishing device.
  • the trap device and the forced dispersion device may be directly connected, and the trapped object trapped using the trap device or dispersed using the forced dispersion device may be used.
  • the dispersion (liquid containing the object to be trapped before trapping) is transferred between both devices, that is, from the trapping device to the forced dispersion device, or through a transfer means for transferring the dispersed material from the forced dispersion device to the trapping device. May be indirectly linked.
  • a separation device having a structure in which a forced dispersion force is applied to a container holding a trap filter is exemplified.
  • a structure capable of directly applying a forced dispersing force by a shaking device to a container holding a trap filter and performing trapping by reduced pressure filtration ie, a separation device capable of shaking the entire reduced pressure filtration device.
  • Examples of the separation device in which the trap device and the forced dispersion device are indirectly connected via the transfer means include, for example, transfer of a tube or pipette for transferring the trapped trapped object or the dispersion itself.
  • a separating device in which the trap device and the forced dispersion device are indirectly connected by means, or by means of transferring the trap device itself to the forced dispersion device or by means of transferring the forced dispersion device itself to the trap device, And a separation device connected to the device.
  • the separation device of the present invention having such a configuration, it is possible to trap the trap target from the mixture by the trap device and disperse the trapped trap object in the liquid by the forced dispersion device. it can.
  • the trap target is trapped from the mixture. You can also.
  • the separation device of the present invention can be automated by an automatic control device, and the target can be efficiently separated. That is, the separation device of the present invention may further include a trap device, a forced dispersion device, and optionally a control device for controlling each operation of the transfer device so as to be operated according to a predetermined operation protocol. Is within the range.
  • the target object that can be separated by trapping a trap target from a mixture by the trap device and dispersing the trapped trap object in a liquid by the forced dispersion device includes, for example, intracellular Examples include nucleic acids, proteins, antibodies and the like.
  • the liquid containing the trap target before trapping Target substances that can be separated by trapping the trapping object from the mixture after sufficiently trapping the trapping object before trapping by subjecting the trapping object to the forcible dispersion device in advance include, for example, gels after electrophoresis. Examples include nucleic acids, proteins in the gel after electrophoresis, and peptides in the gel after electrophoresis.
  • the target object which can be separated by trapping the trapping object from the mixture while simultaneously dispersing the trapping object before trapping by supplying the liquid containing the trapping object before trapping to the aforementioned forced dispersion device in advance includes intracellular Examples include nucleic acids, proteins, antibodies and the like.
  • the second invention relates to a method for separating a biological substance using the separation device of the first invention.
  • the separation apparatus can be used, for example, for purifying biological substances (for example, nucleic acids, proteins, antibodies, etc.).
  • An example of protein purification is a batch process using an insoluble carrier and a protein solution used for protein purification.
  • the separation apparatus of the present invention can be suitably used for the batch process. That is, the protein solution and the insoluble carrier for protein purification are added to the trap container of the separation device of the present invention, and then the insoluble carrier for protein purification is dispersed in the protein solution by the forced dispersion device of the separation device of the present invention. It is possible to promote the adsorption of the substance adsorbed on the insoluble carrier to the carrier.
  • the insoluble carrier for protein purification examples include resins generally used for protein purification, for example, ion exchange resins, affinity chromatography resins, hydrophobic chromatography resins, and the like.
  • the separation device of the present invention can also be used for buffer exchange of a protein solution. For example, by attaching a fractionation membrane generally used for protein purification capable of trapping a target protein to a trap container of the separation device of the present invention, adding a target protein solution to the container, and performing a filtration operation. The protein in the solution can be concentrated. Next, a buffer solution having a composition to be exchanged is added to the trap container, the concentrated protein is diluted, and the filtration operation is performed again.
  • the buffer operation of the protein solution can be exchanged by repeating the filtration operation.
  • the liquid in the trap container is sufficiently dispersed and mixed by a forced dispersion device.
  • the exchange efficiency of the buffer component can be increased.
  • the separation device of the present invention can be used for exchanging a buffer component of a nucleic acid solution and the like.
  • the separation device can be used for a method for separating and purifying nucleic acids.
  • the third invention of the present invention comprises: (a) a step of lysing cells and forcibly dispersing a cell lysate to obtain a crude nucleic acid solution;
  • step (c) a step of eluting the nucleic acid from the nucleic acid adsorbent having the nucleic acid trapped in the step (b) under forced dispersion
  • step (a) a method for separating and purifying nucleic acid.
  • the cells in step (a) are preferably dissolved under forced dispersion.
  • the forced dispersion device used in the separation device of the present invention can be used similarly. That is, forced dispersion can be performed by using a shaking device, an ultrasonic generator, a publishing device, or the like.
  • forced dispersion can be performed by using a shaking device, an ultrasonic generator, a publishing device, or the like.
  • elution under forced dispersion includes a mode in which forced dispersion is performed together with a dissolution, trap, or elution operation, and a mode in which forced dispersion is performed after a dissolution, trap, or elution operation. This improves the efficiency of lysis, trapping and elution.
  • the separation device by using the separation device according to the first aspect, it is possible to recover high-purity nucleic acid more quickly and easily.
  • the separation apparatus of the present invention can be used.
  • the trap fill used in the cell lysis step is preferably a membrane capable of holding a nucleic acid-holding microorganism or cell ⁇ , or a disrupted state of the microorganism or cell, in an appropriately dispersed state. It is desirable to be able to separate the microorganisms and destroy the separated microorganisms or cells.
  • a porosity glass fiber membrane having pores having a retention particle diameter of 0.3 to 1.0 m can be used, but the ability to retain microorganisms or cells, microorganisms or cells containing nucleic acids
  • a membrane having a pore size of 0.4 to 0.8 m is preferred from the viewpoint of the separation time of the internal components and the like.
  • a membrane having a pore size in this range may be selected according to the sample.
  • the diameter of the retained particles may be uniform or may be distributed in the range of 0.4 to 0.8 ⁇ m.
  • the film area, film thickness, and the like may be selected according to the properties of the sample, the processing amount, the preferable processing time, and the like.
  • a fractionation membrane that can be fractionated by a difference in molecular weight
  • a dialysis method is used for final separation of nucleic acids and other substances, but when the method is applied to the separation apparatus of the present invention, a separation method is used. This can be performed by injecting the solution of the final step into a trap container using an image membrane as a trap filter, and repeating concentration by vacuum filtration and dilution with a nucleic acid storage solution.
  • the fractionation size of the fractionation membrane may be appropriately selected depending on the desired molecular weight of the nucleic acid.
  • the amount of the molecule varies depending on the species of the microorganism, but a trap filter equipped with a filter having a fraction size of 500,000 or more may be used.
  • the fractionation membrane is preferably a membrane that adsorbs a small amount of nucleic acid.
  • a YM-based (cellulose) filter manufactured by Amicon can be suitably used.
  • a water-insoluble nucleic acid-adsorbing carrier capable of selectively adsorbing nucleic acids in the presence of chaotropic ions can be used.
  • the shape of the nucleic acid adsorption support is not particularly limited, and may be a particle or a fiber.
  • the particulate nucleic acid-adsorbing carrier include silica gel particles and glass powder. From the viewpoint of physical stability of the target nucleic acid, the particle diameter is preferably in the range of 2 to 10 m. like this
  • the particulate nucleic acid adsorbent for example, silica gel (manufactured by Fuji Syril) and glass powder in EASYTRAP Ver.
  • the trap fill for holding the nucleic acid adsorption carrier is preferably a membrane having a pore size of 0.5 / m to l / m in order to prevent clogging.
  • the membrane is preferably a membrane that adsorbs a small amount of nucleic acid when eluting the nucleic acid from the nucleic acid adsorbent, and examples thereof include a filter made of polytetrafluoroethylene.
  • a fiber-shaped nucleic acid adsorbent if it is formed in a filter shape, it can be used as a filter for a trap container.
  • the filter for example, a GFZC filter manufactured by Whatman can be used.
  • a chaotropic substance is a substance that has the effect of destroying the solvent structure of water and suppressing a decrease in entropy of water that occurs when a hydrophobic substance comes into contact with water.
  • a chaotropic ion is an ion having the above properties. is there.
  • nucleic acids are adsorbed on nucleic acid adsorbents such as glass depending on the ion species used.
  • the effective concentration is also different, for example, when silica gel is used,
  • guanidine hydrochloride is dissolved at 3M or more, and sodium iodide is dissolved at 3M or more.
  • step (D) transferring the crude nucleic acid solution obtained in the step (C) to a trap device for a nucleic acid separation step in which a nucleic acid adsorbent is held,
  • step (F) a step of separating the nucleic acid adsorbent obtained in the step (E) and a liquid component
  • (G) a step of injecting the nucleic acid elution solution into the trap device for the nucleic acid separation step, and eluting the nucleic acid from the nucleic acid adsorbent obtained in the step (F).
  • the forced dispersion device of the present invention When performing the steps (B), (C), (E) and (G) in the method for separating and purifying nucleic acids, the forced dispersion device of the present invention imparts a forced dispersion force to the contents in the trap device. By uniformly dispersing the nucleic acids, the efficiency of nucleic acid separation and purification is significantly improved.
  • the trap container for example, when a device having a rotary shaking mechanism is used as the forced dispersion device, for example, the trap device is shaken at an amplitude of 2 mm to 3 mm and 720 rpm.
  • a microbe holding plasmid DNA using the separation device of the present invention having a trap container suitable for vacuum filtration, a trap device using vacuum filtration, and a shaking device.
  • the method of purifying plasmid DNA from the product will be described more specifically according to the method of Carter et al.
  • E. coli JM1 09ZPUC 1 Microorganism containing a plasmid DNA, eg E. coli for holding the plasmid pUC l 9 [Eshiwerishia coli (Escherichia coli)] JM1 09 strains (hereinafter, referred to as E. coli JM1 09ZPUC 1 9) culturing, for example, OD 60.
  • E. coli JM1 09ZPUC 1 9 culturing, for example, OD 60.
  • the container shown in FIG. 1 can be used.
  • 1 is a cellulose mixed ester membrane (manufactured by Millipore) having a pore size of 0.45 ⁇ m
  • 2 is a polypropylene vessel.
  • This container was named the first trap container.
  • the first trap container holding a cellulose mixed ester membrane with a pore size of 0.45 ⁇ m, approximately 2.5 ml of the culture solution can be filtered under reduced pressure in approximately 15 minutes, and the cultured cells are trapped. Retained on the filter.
  • (B) a step of injecting a cell suspension solution into the trap device for the cell lysis step, and suspending the cells trapped in the trap device in the step (A).
  • the required amount of cell suspension solution in the first trap container for example, 1 OmM EDTAZ5 OmM Tris-HCl (pH 7.5) Inject 3001 and resuspend by shaking (720 rpm) for at least 15 minutes.
  • the trapped cells are uniformly dispersed in the cell suspension, and the efficiency of cell lysis in step (C) is improved.
  • a cell lysis solution for example, 0.2N
  • a cell lysis solution for example, 0.2N
  • a neutralizing solution for example, 2.5 M potassium acetate (pH 4.8)
  • pH 4.8 potassium acetate
  • the cells are lysed and the chromosomal DNA and protein are denatured and insolubilized. Due to the forced dispersing force of the forced dispersion device, lysis of cells and insolubilization of chromosomal DNA and protein proceed efficiently, so that the yield and purity of the recovered plasmid DNA are improved.
  • sodium hydroxide is hereinafter abbreviated as NaOH and sodium lauryl sulfate is abbreviated as SDS.
  • step (D) a step of transferring the crude nucleic acid solution obtained in the step (C) to a trap device for a nucleic acid separation step holding a nucleic acid adsorbent
  • the processing liquid containing the nucleic acid is recovered from the first trap container into the trap container for the nucleic acid separation step previously set by the decompression operation.
  • the substance insolubilized in step (C) and the crude nucleic acid solution can be separated.
  • the container shown in FIG. 2 can be used as the trap container for the nucleic acid separation step.
  • 3 is a polytetrafluoroethylene membrane (manufactured by Advantech) having a pore diameter of 1.0 / m
  • 4 is a polypropylene vessel.
  • This container was named the second trap container.
  • a nucleic acid adsorbent suspension for example, 5 OmgZm 1 silica gel particles Z7M
  • guanidine hydrochloride ZSmM EDTA 1 OmM Tris-HCl pH 7.5
  • a nucleic acid-adsorbing carrier in the form of a filter for example, a trap container in which the polytetrafluoroethylene membrane of the container shown in FIG. 2 is replaced with a glass filter can be used.
  • the container contains a chaotropic substance in advance. It is preferable to add an appropriate amount of a buffer, for example, 7 M guanidine hydrochloride Z2 mM EDTA / 10 mM Tris-HCl (pH 7.5).
  • step (E) contacting the nucleic acid adsorbent with the crude nucleic acid solution transferred in step (D) in a trap device for a nucleic acid separation step
  • the second trap container is shaken at 72 Orpm for about 5 minutes to sufficiently mix the internal solution. This promotes the adsorption action (trap) between the silica gel particles and the nucleic acid.
  • the nucleic acid is adsorbed (trapped) on the glass filter by passing the crude nucleic acid solution through the glass filter by a decompression operation. Shake the trap container for about 1 minute before depressurizing, and thoroughly mix the internal solution to promote the adsorption action (trap) between the glass fill and the nucleic acid.
  • this operation and the following step (F) are performed simultaneously.
  • step (F) a step of separating the liquid component from the nucleic acid adsorbent obtained in the step (E).
  • the nucleic acid adsorbent holding carrier that has adsorbed the nucleic acid by the decompression operation is filtered. Trap on.
  • the nucleic acid and other impurities in the crude nucleic acid solution obtained in the step (D) can be separated.
  • a washing solution containing an appropriate amount of ethanol in a trap container holding the nucleic acid adsorbent obtained by the above method preferably, the final concentration of ethanol is 40% (V / V) or more
  • 50% ethanol (VZV ) Z10 OmM sodium chloride 2.5mM EDTA / l OmM Tris-hydrochloric acid (pH 7.5) (hereinafter referred to as silica washing solution) is added, and the nucleic acid adsorbent is washed by discarding the liquid by depressurization. .
  • the washed nucleic acid adsorbent is dried by decompression for about 2 minutes.
  • an appropriate amount of eluate for example, sterile water or TE solution (10 mM Tris-HCl (pH 8.0) Ilm M EDTA) is added to the second trap container, and the plasmid DNA is removed from the second trap container by decompression. Collect the eluate in a container, for example, an Eppendorf tube.
  • silica gel particles are used as the nucleic acid adsorbent, it is possible to improve the nucleic acid recovery rate by adding a shaking operation to this step.
  • the nucleic acid obtained by the above operation can be subjected to various genetic engineering techniques.
  • the DNA obtained by the above method can be cleaved by a restriction enzyme, and can be used as a type II nucleic acid amplification method represented by PCR (Polymerase Chain Reacion).
  • RNase treatment may be added before step (E), for example, step (C) of the above method.
  • step (E) the yield of nucleic acid is improved by adding an enzyme that dissolves the cell wall specific to the cells to the cell suspension.
  • lysozyme may be added to a cell suspension to a final concentration of 4 mgZm1.
  • an antifoaming agent may be used as necessary.
  • the antifoaming agent include Adekinol (Asahi Denka Kogyo), Ainol (Manufactured by Biot), Nissan Day Home CB 442 (manufactured by NOF Corporation), etc. What is necessary is just to add an enol.
  • foaming and scattering of the filtrate can be prevented. For example, by adjusting the degree of vacuum to around -65 OmmHg in the presence of 0.05% Ainol, foaming can be completely prevented. Can be suppressed.
  • the flow through the filtration membrane is improved in addition to the defoaming action.
  • the step (E) when DNA is adsorbed to silica gel in the presence of 3.5 to 4.5 M guanidine hydrochloride in the second trap container described in this specification, ethanol having a final concentration of 20% (V / V) is added. Then, the amount of DNA adsorbed is almost the same as when ethanol is not added, and the flow time at a vacuum of about 65 OmmHg can be reduced from about 12 minutes to about 6 minutes.
  • the load of microorganisms or cells containing plasmid DNA per fill area of the trap container is high, for example, if a porous silicate glass fiber membrane with pores with a retention particle diameter of 0.8 / m is used, good.
  • a porous silica glass fiber membrane manufactured by Advantech: GA-200
  • the above E. c 0 1 i JM 109 / pUC 9D 6 of 1 9. about 1.5 ml of the culture solution of about 6 can be filtered under reduced pressure in about 3 minutes, and a separated solution containing plasmid DNA can be collected in about 5 minutes.
  • the purification of plasmid DNA by the above steps (A) to (G) using the particulate nucleic acid adsorbent is carried out in the cell lysis step trap vessel (first trap vessel) shown in FIG.
  • first trap vessel first trap vessel
  • second trap container second trap container
  • sample and reagent transfer means and automatic control means shown in Fig. 3
  • the cell suspension solution 301 is transferred into the first trap container in which the cells of the step (2) are trapped, and added.
  • the first trap container is shaken by a forced dispersion device, and the trapped cells are suspended in the cell suspension in the step (3).
  • the neutralizing solution 20001 is transferred to the first trap container after cell lysis in the above step (6) and added.
  • the first trap container is shaken by a forced dispersion device, and neutralization with the neutralizing solution added in the step (7) is performed under forced dispersion.
  • the second trap container is shaken by a forced dispersion device, and the nucleic acid in the filtrate obtained in the step (10) is adsorbed to the nucleic acid adsorbent under forced dispersion.
  • the nucleic acid adsorbent having the nucleic acid adsorbed in the step (11) is trapped on the filter membrane surface by vacuum filtration.
  • the washing solution 500/1 is transferred and added to the second trap container in which the nucleic acid adsorbent of the step (12) is trapped.
  • the second trap container is shaken by the forced dispersion device, and the nucleic acid adsorbent is mixed with the washing solution transferred in the step (13) under the forced dispersion to wash the nucleic acid adsorbent.
  • step (14) Adsorption of nucleic acid washed in step (14) on the filter membrane surface by vacuum filtration Trap the body.
  • the washing solution 500/1 is transferred and added to the second trap container in which the nucleic acid adsorbent has been trapped in the step (15).
  • the second trap container is shaken by a forced dispersion device, and the nucleic acid adsorbent is mixed with the washing solution transferred in the step (16) under forced dispersion to wash the nucleic acid adsorbent.
  • the method for separating and purifying nucleic acids comprising the above steps (A) to (G) is suitable for purifying circular DNA such as plasmid DNA, but it is not suitable for whole blood, which is frequently performed in the clinical field, etc. It is not suitable for extracting chromosomal DNA from cultured cells. Even in the case of such a linear DNA, the nucleic acid can be more easily extracted and purified by using the apparatus of the present invention in the following steps.
  • a nucleic acid separation device including the trap container for nucleic acid separation step (second trap container) shown in FIG. 2 and the separation device, sample and reagent transfer means and automatic control means shown in FIG. It is also possible to do this.
  • the nucleic acid used in the following step is preferably a cell covered with only the plasma membrane, and includes, for example, white blood cells contained in blood.
  • detergents such as plant cells and microbial cells, which cannot lyse cells with SDS alone Even so, there is no problem as long as the cell wall has been removed. In this case, the cell wall does not need to be completely removed, but it is sufficient that the cell wall is removed to the extent that the cells burst in a hypotonic solution.
  • a trap device for the cell lysis step is not necessary, and only a trap device for the nucleic acid separation step is required.
  • a granular nucleic acid adsorption carrier is used, the following embodiments are exemplified.
  • step (iv) a step of injecting the nucleic acid eluate into the trap device for the nucleic acid separation step, and eluting the nucleic acid from the nucleic acid adsorbent obtained in the step (iii);
  • An extracorporeal sample containing human blood cells for example, 100/1 human whole blood subjected to a known anticoagulation treatment is injected into the second trap container, and then a diluting reagent is used to dilute the extracorporeal sample.
  • a diluting reagent is used to dilute the extracorporeal sample.
  • inject the required amount of cell lysis solution eg, 150/1 for 0.2% SDS, and lyse the cells by shaking (1700 rpm) for at least 5 minutes.
  • step (ii) in the step of bringing the cell lysate into contact with the nucleic acid adsorbent in step (ii), the efficiency of selective adsorption of nucleic acids contained in the cell lysate to the nucleic acid adsorbent increases.
  • step (ii) a step of injecting the nucleic acid adsorbent suspension into the trap device for the nucleic acid separation step, and bringing the cell lysate obtained in the step (i) into contact with the nucleic acid adsorbent.
  • the cell lysate obtained in step (i) may be mixed with a nucleic acid adsorbent suspension containing a nucleic acid adsorbent and a chaotropic substance solution.
  • Omg / m 1 Silica gel particles 7M guanidine hydrochloride / 2mM EDTA In the case of OmM Tris-HCl (pH 7.5) Inject 7001.
  • the blood cells are lysed and the extracted nucleic acid is adsorbed on the silica gel particles.
  • the efficiency of cell lysis and the efficiency of nucleic acid adsorption to silica gel particles are improved.
  • an appropriate organic solvent is added to the nucleic acid adsorbent suspension, for example, in the case of the exemplified nucleic acid adsorbent suspension, ethanol is added to a final concentration of 40% (V / V).
  • the amount of DNA adsorbed on silica gel hardly changes, and the time required for filtration in the separation of the nucleic acid adsorbent and the liquid component in step (iii) is reduced. It can be shortened.
  • the cell lysate obtained in step (i) may be mixed with a chaotropic substance solution, for example, 7 M guanidine hydrochloride Z2 mM EDTAZl OmM Tris-HCl (pH 7.5 In the case of), blood cells are lysed by injecting 700/1. Performing the shaking operation at this point improves the lysis efficiency of the cells.
  • a vacuum operation is performed, and the nucleic acid is adsorbed on the glass filter by passing the cell lysate through the glass filter.
  • an appropriate organic solvent for example, ethanol having a final concentration of 40% (V / V) to the nucleic acid adsorbent suspension. This operation also serves as part of the next step (iii).
  • step (iii) Step of separating the nucleic acid adsorbent obtained in step (ii) from the liquid component
  • the silica gel particles are then placed on the filter of the second trap container by a decompression operation. To trap. By this step, nucleic acids and other intracellular impurities can be separated.
  • a step of washing the nucleic acid adsorbent is added in this step, the purity of the obtained nucleic acid can be further improved.
  • the degree goes up.
  • V / V 50% ethanol
  • the washing action can be further enhanced by adding a shaking operation during this washing.
  • a washing solution containing an appropriate amount of ethanol as in the case of using silica gel particles in the filter to which the nucleic acid obtained in step (ii) is adsorbed is trapped.
  • the nucleic acid adsorbent is washed by passing a washing solution through a filter by a vacuum operation.
  • the washed nucleic acid adsorbent is dried by decompression for about 2 minutes.
  • an appropriate amount of eluate for example, sterile water, TE solution (10 mM Tris-HCl (PH8.0) I ImM EDTA) is added, and then the eluate containing nucleic acid is removed from the second trap container by a reduced pressure operation. Collect in a container such as an Eppendorf tube.
  • silica gel particles are used as the nucleic acid adsorbent, the recovery rate of nucleic acids can be improved by adding a shaking operation to this step.
  • the washed nucleic acid adsorbent is dried by a reduced pressure operation for about 2 minutes.
  • an appropriate amount of eluate for example, sterile water or TE solution (10 mM Tris-HCl (pH 8.0) I ImM EDTA)
  • the eluate containing the nucleic acid is placed in a second trap container by depressurization, and the eluate is placed in a container, for example, an ETPEN. Collect in Dorf tube.
  • RNase treatment can be performed before contacting the cell lysate with the nucleic acid adsorbent to obtain higher purity DNA with lower RNA content. it can. For example, by using a material containing 13 ⁇ 4 Na se A final concentration of 1 00 / / 1) 11 to the reagent for diluting, it is possible to perform the degradation of RNA at the same time in step (i).
  • the DNA obtained by the above operation can be cleaved with a restriction enzyme, and can be used, for example, as a type II nucleic acid amplification method represented by PCR.
  • the DNA obtained by the above procedure has a purity that can be used as type II of LA PCR (Long and Accurate PCR), which is known to inhibit the reaction with low-purity type II DNA. have.
  • a circular double-stranded plasmid DNA is prepared from microbial cells by the method for separating and purifying nucleic acids according to the above steps (i) to (iv), a cell wall removing operation, a step ( By performing genomic DNA and RNA separation operations after iv), highly pure circular double-stranded plasmid DNA having a low protein content and low RNA content can be obtained.
  • the cell wall removal operation can be performed by suspending Escherichia coli in lysozyme-containing 5 OmM Tris-HCl buffer (pH 7.5) and allowing to stand at room temperature for 5 minutes.
  • Genomic DNA and RNA separation is performed, for example, by adding 2 volumes of 0.2N NaOH / 1.0% SDS solution to 1 volume of nucleic acid solution containing circular double-stranded plasmid DNA, mixing, and leaving the mixture on ice for 5 minutes. A degradation and denaturation of genomic DNA are performed. Thereafter, 1.5 volumes of 2.5 M potassium acetate (pH 4.8) is added, mixed, and left in ice for 10 minutes to insolubilize genomic DNA. The solution is centrifuged at 14000 g for 10 minutes to precipitate and remove denatured genomic DNA. By adding ethanol to the obtained supernatant and precipitating plasmid DNA, high-purity double-stranded plasmid DNA can be obtained.
  • 2.5 M potassium acetate pH acetate
  • the trap device of the present invention employs a reduced-pressure filtration mechanism for treating a large number of samples, thereby shortening the time. In addition, a number of sample processes can be performed.
  • the method for separating and purifying nucleic acids using the apparatus of the present invention is not limited to the above method.
  • steps (A) to (G) when purifying nucleic acid from a reaction solution in which an enzymatic reaction has been performed, in the method for separating and purifying nucleic acid comprising steps (A) to (G), steps (A) to (C) are unnecessary, and D) to (G) may be performed.
  • the reaction solution after the enzymatic reaction may be transferred in place of the crude nucleic acid solution obtained in the step (C) in the step (D).
  • the method for separating and purifying nucleic acids by the steps (i) to (iv) is not limited to the method for separating and purifying nucleic acids present in cells, for example, the method of recovering DNA from agarose gel and the method of separating phage particles based on Christensen et al. It can be applied to the recovery of DNA. That is, an agarose gel or phage particle suspension containing a nucleic acid is used in place of the cell suspension in the above step, and the cell lysis step and separation of the nucleic acid and intracellular impurities are simultaneously performed. Recovery of DNA may be performed according to the method.
  • the reagent used in each step is a reagent suitable for RNA purification, for example, RNa It is necessary to use a reagent that has been confirmed to be sefree.
  • the fourth invention provides a kit for separating and purifying nucleic acids by the method of the third invention, which contains at least a substance having a nucleic acid-adsorbing ability.
  • the kit comprises, as an essential component, a substance having a nucleic acid adsorption ability as described above.
  • a reagent for extracting nucleic acid from cells such as a cell lysis solution
  • silica gel particles can be used as the substance having nucleic acid adsorption ability
  • SDS solution can be used as the cell lysis solution.
  • a high purity DNA separation kit can be obtained by including an RNA degrading enzyme (RNase) for decomposing contaminants.
  • RNase RNA degrading enzyme
  • a surfactant for cell lysis and protein coagulation may be included in the kit.
  • a cell lysis step trap container having the structure shown in FIG. 1 was prepared.
  • the container had a structure suitable for vacuum filtration.
  • a trap container for a nucleic acid separation step having the structure shown in FIG. 2 was prepared.
  • the container had a structure suitable for vacuum filtration.
  • FIG. 3 is a schematic diagram of the device.
  • reference numeral 5 denotes a trap container
  • 6 denotes a trap container fixing device
  • 7 denotes a rotary shaking device
  • 8 denotes a decompression generator
  • 9 denotes a waste liquid receiving tray.
  • E. coli JM109 transformed with plasmid pUC19 was added to 5 LB medium containing ampicillin (10 g tryptone, 5 g yeast extract, 5 g sodium chloride liter) 2. Inoculate 2.5 ml and culture temperature 37. C ⁇ Incubate for 16 hours at a shaking speed of 160 rpm and OD 6 . . About 6 cultures were prepared.
  • Step 3 Step of mixing the crude nucleic acid solution and the nucleic acid adsorbent suspension obtained in Step 2 above.
  • + added after each numeral indicates that the shaking operation was performed in the step. This indicates that the shaking operation was not performed in the step.
  • ⁇ indicates that the filter was clogged during purification.
  • the amount of plasmid DNA recovered when the shaking operation was not added to some of the steps was about one third to about 50 minutes when the shaking operation was added to all the steps.
  • the shaking operation in the cell suspension step and the lysis step significantly increased the yield.
  • EcoRI a restriction enzyme that cuts about 0.5 ⁇ g of the DNA prepared in the 1+ 2+ 3+ step in the above (2) at one place in pUC 19 DNA
  • the circular pUC19 DNA was cut at the-site to become linear, and did not inhibit the reaction of the restriction enzyme.
  • the brassmid was made into a ⁇ type, and a dioxygenase was produced by BcaBEST Dideoxy Sequen ci ng Kit (manufactured by Takara Shuzo) using Fluo rescein Primer M4 (manufactured by Takara Shuzo) as a fluorescent primer.
  • BcaBEST Dideoxy Sequen ci ng Kit manufactured by Takara Shuzo
  • Fluo rescein Primer M4 manufactured by Takara Shuzo
  • Escherichia coli HB101 transformed with pBR322 was cultured in the same manner as in Example 2, and PBR322 was purified in the same manner as in Example 2.
  • pBR322 purified in the 1 + 23 + step was cut with 10 units of restriction enzyme BamHI (manufactured by Takara Shuzo Co., Ltd.) at 30 for 1 hour to obtain a favorable cleavage pattern. Obtained from electrophoresis results.
  • Injected human cultured cells were washed with PBS (HL- 6 0 (ATCC CCL- 24 0)) 1 0 0/1 (1 X 1 0 4 ⁇ 1 X 1 0 6 cells) to the second trap vessel,
  • Example The cell suspension containing RNase A described in 2 (2) was added to 1001 and 0.2% SDS solution at 200/1, and shaken for 5 minutes. Thereafter, 500/1 of the dissolved nucleic acid adsorbent suspension described in (2) of Example 2 was added thereto, and mixed for 5 minutes by shaking.
  • the second trap container was depressurized for 3 minutes and 30 seconds to trap the gel particles, and the washing solution 3001 described in (2) of Example 2 was added, and the mixture was shaken for 4 minutes to remove the silica gel particles.
  • Washing was performed. After reducing the pressure for 2 minutes to trap the silica gel particles and performing the washing treatment again, a washing solution of 501 was added and washing was performed under reduced pressure for 2 minutes. Add 1501 sterile water to the silica gel, shake for 15 minutes to elute the DNA from the silica gel particles, and finally depressurize for 2 minutes to transfer the eluate containing DNA to an Eppendorf tube. Collected. As a result, about 4 g of DNA was obtained.
  • the absorbance at a wavelength of 28 O nm, which is mainly close to the maximum absorption wavelength of the protein (OD 28 ), and the absorbance of the wavelength at 26 O nm, which is mainly close to the maximum absorption wavelength of the nucleic acid (OD 26 ) was measured.
  • the ratio (OD 26 / OD 28 ) was calculated to be 1.85.
  • DNA was purified using the SDS cell lysis method.
  • Three kinds of known anticoagulation treatments that is, human whole blood 801 treated with citrate, EDTA and heparin are injected into the second trap container, and the cells described in (2) of Example 2 containing RNase 100/1 of a suspension reagent and 1501 of a 0.2% SDS solution were added, and shaking operation at 720 rpm was performed for 3 minutes.
  • 7001 of the nucleic acid adsorbent suspension prepared in the same manner as in (2) of Example 2 was added thereto, and the mixture was shaken at 720 rpm for 5 minutes. The liquid containing more impurities was discarded and silica gel particles were trapped.
  • Example 5 the nucleic acid sample obtained in (1) of Example 5 was subjected to various genetic engineering means. First, about 0.2 ⁇ g of each of the obtained nucleic acids was digested with the restriction enzyme EcoRI in the same manner as in (3) of Example 2, and as a result, DNA was digested. Therefore, in Example 5,
  • the nucleic acid sample obtained in (1) of Example 5 was designated as type III, and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing was used as a primer pair to form TaKaRa PCR Amp. lificati on K it (Takara Shuzo Co., Ltd. :) or LA PCR TM kit Ver. 2 (Takara Shuzo) to perform a nucleic acid amplification reaction.
  • the number of PCR cycles is 25 when TaKaRa PCR Amp kit is used, and 30 when LA PCR TM kit Ver. 2 is used.
  • the resulting amplified DNA fragment was confirmed by agarose gel electrophoresis.
  • the nucleic acids in the agarose gel were detected by staining the agarose gel with EtBr and irradiating it with ultraviolet light of 254 nm.
  • the expected DNA fragment having a length of about 410 bp was obtained without any inhibition of the amplification reaction by the nucleic acid amplification reaction using any nucleic acid sample as type III.
  • the LA PCR TM kit Ve using the nucleic acid obtained in (1) of Example 5 as type III and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing as a primer pair A nucleic acid amplification reaction according to r.2 was performed. The number of PCR cycles is 25.
  • Plasmid DNA purification kit (100 times)
  • the separation device of the present invention enables simple and rapid purification of biological materials such as nucleic acids, proteins, and peptides. Sequence listing
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)

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Abstract

L'invention concerne un appareil permettant d'isoler un objet de piégeage d'un mélange. L'appareil est caractérisé par le fait qu'il comporte un dispositif servant à piéger l'objet contenu dans un mélange, et un dispositif de dispersion par contrainte permettant d'appliquer une force de dispersion par contrainte à l'objet piégé par le dispositif de piégeage et/ou à un liquide contenant l'objet avant piégeage; un procédé permettant d'isoler une substance biologique au moyen de l'appareil d'isolement; un procédé d'isolement et de purification d'acides nucléiques, caractérisé par le fait qu'il comporte les étapes consistant à (a) dissoudre les cellules et disperser par contrainte les cellules dissoutes pour préparer une solution brute d'acides nucléiques, (b) piéger les acides nucléiques dans la solution brute d'acides nucléiques obtenue à l'issue de l'étape (a) à l'aide d'un adsorbant d'acides nucléiques sous dispersion par contrainte, et (c) éluer les acides nucléiques piégés à l'étape (b) de l'adsorbant d'acides nucléiques sous dispersion par contrainte; et une trousse permettant d'isoler et de purifier les acides nucléiques, utile dans le procédé mentionné. Des substances biologiques telles que des acides nucléiques, des protéines et des peptides peuvent être purifiées aisément et rapidement à l'aide de l'appareil de séparation, du procédé d'isolement, du procédé et de la trousse d'isolement et de purification d'acides nucléiques de la présente invention.
PCT/JP1998/001455 1997-03-31 1998-03-30 Appareil d'isolement WO1998043724A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2355717A (en) * 1999-10-28 2001-05-02 Amersham Pharm Biotech Uk Ltd DNA isolation method
JP2004519234A (ja) * 2000-10-31 2004-07-02 ヒタチ ケミカル リサーチ センター インコーポレイテッド 核ｍＲＮＡの収集および使用方法
JP2007124952A (ja) * 2005-11-04 2007-05-24 Hitachi High-Technologies Corp 核酸精製方法及び核酸精製器具
KR20110072276A (ko) * 2009-12-22 2011-06-29 삼성전자주식회사 핵산 분리 방법 및 장치
JP2018004651A (ja) * 2007-10-24 2018-01-11 バイオマーカー ストラテジーズ リミテッド ライアビリティ カンパニー 細胞分析の進歩した方法および装置

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JP5729904B2 (ja) 2009-06-02 2015-06-03 キヤノン株式会社 細胞から蛋白質、ｄｎａ、ｒｎａを調製する方法
JP6113083B2 (ja) * 2011-02-21 2017-04-19 リーアニクス・インコーポレイテッドＲｈｅｏｎｉｘ， Ｉｎｃ． マイクロ流体素子を基礎とした核酸精製方法
JP6771161B2 (ja) * 2016-03-10 2020-10-21 パナソニックＩｐマネジメント株式会社 核酸抽出装置

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JPS6041576A (ja) * 1983-04-25 1985-03-05 ア−／エス　エン．フオス　エレクトリツク 流体やプラスチツク材または固形材の物質、または粒子状生成物の産出または遊離及び分離の装置及び方法とその装置の使用法
JPH0242969A (ja) * 1988-07-30 1990-02-13 Sekisui Chem Co Ltd 生物学的試料の処理装置
JPH03105251A (ja) * 1989-09-20 1991-05-02 Hitachi Ltd 試料調製装置
JPH04181163A (ja) * 1990-11-14 1992-06-29 Toray Eng Co Ltd 臨床検査前処理装置
JPH0947278A (ja) * 1995-08-04 1997-02-18 Tomy Seiko:Kk Ｄｎａ抽出精製方法及び装置

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JPS58158345U (ja) * 1982-04-16 1983-10-22 紀本電子工業株式会社 汚濁水の計測前処理装置
JPS6041576A (ja) * 1983-04-25 1985-03-05 ア−／エス　エン．フオス　エレクトリツク 流体やプラスチツク材または固形材の物質、または粒子状生成物の産出または遊離及び分離の装置及び方法とその装置の使用法
JPH0242969A (ja) * 1988-07-30 1990-02-13 Sekisui Chem Co Ltd 生物学的試料の処理装置
JPH03105251A (ja) * 1989-09-20 1991-05-02 Hitachi Ltd 試料調製装置
JPH04181163A (ja) * 1990-11-14 1992-06-29 Toray Eng Co Ltd 臨床検査前処理装置
JPH0947278A (ja) * 1995-08-04 1997-02-18 Tomy Seiko:Kk Ｄｎａ抽出精製方法及び装置

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2355717A (en) * 1999-10-28 2001-05-02 Amersham Pharm Biotech Uk Ltd DNA isolation method
JP2004519234A (ja) * 2000-10-31 2004-07-02 ヒタチ ケミカル リサーチ センター インコーポレイテッド 核ｍＲＮＡの収集および使用方法
JP2007124952A (ja) * 2005-11-04 2007-05-24 Hitachi High-Technologies Corp 核酸精製方法及び核酸精製器具
JP4699868B2 (ja) * 2005-11-04 2011-06-15 株式会社日立ハイテクノロジーズ 核酸精製方法及び核酸精製器具
JP2018004651A (ja) * 2007-10-24 2018-01-11 バイオマーカー ストラテジーズ リミテッド ライアビリティ カンパニー 細胞分析の進歩した方法および装置
KR20110072276A (ko) * 2009-12-22 2011-06-29 삼성전자주식회사 핵산 분리 방법 및 장치
KR101626846B1 (ko) 2009-12-22 2016-06-02 삼성전자주식회사 핵산 분리 방법 및 장치

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