WO2001011364A1 - Procede d'etiquetage automatique faisant appel a un distributeur automatique, procede de separation automatique d'une substance cible, procede permettant de determiner la sequence de base et systeme de distribution automatique - Google Patents

Procede d'etiquetage automatique faisant appel a un distributeur automatique, procede de separation automatique d'une substance cible, procede permettant de determiner la sequence de base et systeme de distribution automatique Download PDF

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Publication number
WO2001011364A1
WO2001011364A1 PCT/JP2000/005305 JP0005305W WO0111364A1 WO 2001011364 A1 WO2001011364 A1 WO 2001011364A1 JP 0005305 W JP0005305 W JP 0005305W WO 0111364 A1 WO0111364 A1 WO 0111364A1
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WO
WIPO (PCT)
Prior art keywords
genetic material
liquid passage
magnetic particles
labeled
dispenser
Prior art date
Application number
PCT/JP2000/005305
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English (en)
Japanese (ja)
Inventor
Sumihare Noji
Hideji Tajima
Original Assignee
Precision System Science Co., Ltd.
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Filing date
Publication date
Application filed by Precision System Science Co., Ltd. filed Critical Precision System Science Co., Ltd.
Publication of WO2001011364A1 publication Critical patent/WO2001011364A1/fr

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Classifications

    • 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
    • G01N35/028—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 having reaction cells in the form of microtitration plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to an automatic labeling method using a dispenser, a method for automatically selecting a target substance, a method for determining a base sequence, and an automatic dispensing system.
  • the present invention relates to various fields such as genetic manipulation, medical treatment, medicine, physiological hygiene, health, living organisms, food or materials in fields such as agriculture, engineering, pharmacy, medicine, or science such as chemistry or biology. It is used for inspection, measurement, reaction, production, extraction or observation.
  • the operator uses a pipette from a container containing the relevant reagents or the like. Depending on the method, it was necessary to aspirate, transfer, and discharge a predetermined volume to mix the reagents to obtain a reaction solution. In addition, in order to purify the target substance, it was necessary to control the temperature by separating using a centrifugal separator or controlling the temperature of the solution contained in a variable-temperature bath capable of changing the temperature. By the way, in the conventional labeling method, each step is directly controlled and operated by an operator, so that the processing result greatly depends on the presence or absence of the operator's operability.
  • An object of the present invention is to provide a labeling method, a method for automatically selecting a target substance, a method for determining a base sequence, and an automatic dispensing system.
  • the use of a dispenser with a simple structure, compactness, and multifunctional integrated devices enables the use of a dispenser that can perform highly efficient processing without increasing the scale of the device. It is an object of the present invention to provide a chemical conversion method, a method for automatically selecting a target substance, a method for determining a base sequence and an automatic dispensing system.
  • an automatic labeling method using a highly reliable dispenser suitable for handling a small amount of genetic material an automatic sorting method for the target substance, a base sequence determination method, and an automatic dispensing system.
  • a liquid passage a magnetic portion for applying and removing a magnetic field from the outside to the liquid passage, and a pressure control for absorbing and discharging a fluid by controlling a pressure in the liquid passage.
  • a dispensing machine having a container, a moving unit for relatively moving the dispensing machine or the liquid passage and the container, and a container comprising a large number of storage units.
  • a separation step of separating the magnetic particles a separation step of resuspending the separated magnetic particles to generate and elute the labeling-related genetic material fragment, and mixing the eluted labeling-related genetic material fragment with the new magnetic particles.
  • the labeled genetic material fragment is captured by the magnetic particles, and is applied to the inner wall of the liquid passage by applying a magnetic field when the suspension containing the magnetic particles passes.
  • a purification step of purifying the labeling-related genetic material fragment by repeating once or twice or more the process of resuspending the separated and separated magnetic particles to elute the labeling-related genetic material fragment.
  • the term “related genetic material” refers to a genetic material having a specific nucleotide sequence and a genetic material having a certain association.
  • DNA ⁇ RNA having a specific nucleotide sequence it refers to DNA or RNA having a complementary base sequence, or DNA or RNA having a base sequence capable of binding to the DNA or RNA with a predetermined probability.
  • Labeled-related genetic material refers to related genetic material that has been labeled in combination with a labeling material.
  • the “liquid passage” there is a nozzle of the dispenser itself, or Jt ⁇ formed on a chip detachably mounted on the nozzle as shown in the sixth invention.
  • the extraction step comprises mixing a bacterial colony such as Escherichia coli, into which the vector has been introduced, with a DNA extract, solubilizing the DNA extract, and mixing the DNA by mixing magnetic particles.
  • a washing step of washing the magnetic particles and the liquid passage by sucking the washing liquid through the liquid passage, and eluting the vector while the magnetic particles are adsorbed on the inner wall of the liquid passage.
  • Vector includes plasmids, bacteriophages, and the like.
  • the cleavage step is performed through a liquid passage from each of the accommodating portions accommodating the vector containing the cDNA having the specific base sequence and the predetermined reagent.
  • I is a step of moving to a storage section having a temperature function and discharging and cutting the annular vector, wherein the synthesizing step is a DNA polymerase.
  • the label corresponding to the cDNA that has been aspirated from each of the storage sections containing the predetermined reagents including RNA polymerase and the labeling substance via the liquid passage moved to the storage section having a constant temperature function, and discharged and cut to be discharged.
  • the separation step is a step in which a binding substance that specifically binds to the labeled DNA or labeled RNA is added to a reaction solution containing the labeled DNA or labeled RNA.
  • a binding substance that specifically binds to the labeled DNA or labeled RNA is added to a reaction solution containing the labeled DNA or labeled RNA.
  • the suspension is separated by applying a magnetic field when passing the suspension through a liquid passage.
  • the suspension is sucked from the storage part containing the new magnetic particles, transferred to the storage part having a constant temperature function, discharged, and the labeled DNA fragment or labeled RNA fragment is captured.
  • the magnetic particles are adsorbed and separated on the inner wall of the liquid passage, the labeled DNA fragment or the labeled RNA fragment is eluted from the magnetic particles with a predetermined reagent, and only the magnetic particles are adsorbed and separated into the liquid passage.
  • This is a step of purifying the labeled DNA fragment or labeled RNA fragment by discarding.
  • the predetermined reagents in the cleavage step are water, a cleavage buffer and a cleavage enzyme, and the predetermined reagents in the synthesis step are template, water, RNace inhibition Reagent, synthesis buffer, labeling substance and DNA polymerase or DNA polymerase;
  • the binding substance in the separation step is silica gel;
  • the predetermined reagent in the separation step is a buffer such as acetate; Ethanol, the hydrolyzate in the splitting step is an alkaline hydrolysate, the predetermined reagents in the purification step are water, a buffer, and ethanol, and the binding substance is silica gel. It is something that is.
  • the labeling substance is a compound of one of a pair of specifically binding compound pairs such as a fluorescent substance, biotin and avidin, and DIG. Substances, isotopes, or chemiluminescent substances.
  • the liquid passage is detachably provided to the dispenser.
  • a seventh aspect of the present invention provides a liquid passage, a magnetic force portion that applies and removes a magnetic field from the outside to the liquid passage, and a pressure control that controls the pressure in the liquid passage to absorb and discharge the fluid.
  • a target substance is selected using a dispenser having a portion, a moving portion for relatively moving the dispenser or the liquid passage and the container, and a container including a large number of storage portions.
  • Amplification process and amplified multiple related genes Each containing a fragment, a labeled genetic material fragment labeled with one of a pair of compounds having a specific base sequence and specifically binding, and a magnetic particle coated with the other of the pair of compounds;
  • the target related genetic material fragment is captured by the magnetic particles via the labeled genetic material fragment by sucking the suspension I from the part, moving, discharging, and mixing, and passes through the captured magnetic particles.
  • a separation step of selecting a target related genetic material fragment by applying a magnetic field to the liquid passage and separating the target genetic material fragment.
  • the genetic material in the extracting step is RNA
  • the synthesizing step is a step of synthesizing cDNA as the related genetic material using a reverse transcriptase.
  • the labeled genetic material fragment in the capturing step is a labeled DNA fragment or a labeled RNA fragment
  • the compound pair is a labeled compound such as biotin and avidin, DIG, and an antibody specifically binding thereto.
  • the liquid passage is provided detachably with respect to the dispenser.
  • a tenth aspect of the present invention is directed to a liquid passage, a magnetic force portion that applies and removes a magnetic field from the outside to the liquid passage, and a pressure control that controls a pressure in the liquid passage to absorb and discharge the fluid.
  • the base sequence is determined using a dispenser having a portion, a moving portion for relatively moving the dispenser or the liquid passage and the container, and a container including a large number of storage portions.
  • the “SBH method” refers to a technique for determining a base sequence by hybridization using a large number of oligonucleotides. An unknown DNA fragment is paired with millions of oligonucleotide 'probes immobilized on a panel (DNA chip), and the nucleotide sequence of the DNA fragment is determined from the pairing pattern.
  • the genetic material is DNA.
  • the liquid passage is detachably provided to the dispenser.
  • a thirteenth invention is directed to a liquid passage, a storage unit communicating with the liquid passage, a magnetic unit for applying a magnetic field to and removing the liquid passage, and a pressure adjusting unit for adjusting the pressure in the storage unit.
  • Target substance using a dispenser having a dispenser, a moving unit for changing a relative position between the dispenser or the liquid passage and the container, and a container comprising a large number of storage units.
  • the labeling-related genetic material is captured by magnetic particles by moving and discharging into a container having a constant temperature function, and a magnetic field is applied when the suspension passes through the liquid passage.
  • the genetic material is RNA
  • the related genetic material is cDNA
  • the labeling material is a fluorescent material.
  • the liquid passage is detachably provided to the dispenser.
  • a plurality of liquid passages a magnetic force portion for applying and removing a magnetic field from the outside to each of the liquid passages, and controlling the pressure in each of the liquid passages to suction and discharge the fluid.
  • a dispensing machine having a pressure control unit for performing the following steps: a dispensing machine or a moving unit that relatively moves between the dispensing machine or the liquid passage and the container; and the plurality of liquid passages can be simultaneously inserted.
  • a control unit for controlling operations of the dispenser and the moving unit, and the container stores a suspension in which magnetic particles are suspended.
  • An accommodating section accommodating an unused or reusable chip provided with the liquid passage so that the dispenser can be mounted thereon and the chip for disposal detached from the dispenser, and accommodating waste liquid And a dispenser system having an accommodating section.
  • the “container having an accommodation portion provided so that the plurality of liquid passages can be simultaneously inserted” is, for example, a matrix in which the number of rows is equal to the number of rows. And / or the plurality of housings arranged in a
  • the liquid passage includes a storage portion having a width that can be inserted all at once.
  • matrix includes a case in which one row includes a plurality of columns or a plurality of rows includes one column.
  • the moving unit that moves the liquid passage with respect to the container may be included in the dispenser.
  • control unit comprises: setting means for setting the dispenser, the moving unit, and the container; and Or display means for displaying data including the set arrangement of the accommodation sections of the containers.
  • ⁇ settings '' include the setting of various operation procedures by the dispenser, the designation or arrangement of the storage section to be used, the bow I, the presence or absence of discharge, the suction amount, the discharge amount, the suction and discharge speed, Includes presence / absence, temperature setting, incubation time, moving direction, moving distance, etc.
  • control unit is configured to control a type of the liquid to be discharged from the liquid passage of the dispenser, a liquid amount thereof, and a discharge destination storage unit. Based on the state or based on selection by the setting means, control is performed so as to perform ejection in the landing mode, in liquid, or in the air.
  • the “state in the storage section” refers to a state such as whether the storage section is empty, whether the liquid is stored, whether the type of the stored liquid is the same as or different from the type of the liquid to be discharged, and the like.
  • the ⁇ landing mode '' means that the tip of the pipette tip or nozzle is once brought into contact with the inner bottom of the container to check the position of the inner bottom, and then the liquid can be ejected from the tip.
  • the liquid is discharged from the pit tip or nozzle at a position slightly raised from the inner bottom so that the discharged liquid can move to the inner bottom.
  • “In liquid” means that the liquid is discharged while the tip of the pipette tip or nozzle is inserted in the liquid. Thus, even if the amount of liquid to be discharged is minute, the liquid can be reliably transferred to the storage section. “In the air” refers to discharging from the air without putting the tip of the pipe tip or nozzle in the liquid. In this case, the amount of liquid to be discharged is relatively large, and the liquid can be transferred without using the surface tension. This is useful when precision of the liquid amount does not matter. Can be used. In this case, there is an advantage that the bit tip or the nozzle is not contaminated. ADVANTAGE OF THE INVENTION According to this invention, even if it discharges a minute amount of liquid or discharges a large amount of liquid, each can be performed reliably and with high reliability.
  • a liquid passage a magnetic force unit for applying and removing a magnetic field from the liquid passage to the liquid passage, and a pressure absorption in the liquid by controlling the pressure in the liquid passage.
  • a labeling process by using a dispenser having a pressure control unit for performing I and discharge, a moving unit for relatively moving between the dispenser and the container, and a container having a large number of storage units. The burden on the operator can be reduced by consistently automating the process of selecting the target substance and determining the base sequence.
  • highly efficient processing can be performed without increasing the scale of the device by using a device having a simple structure and a compact but multifunctional and integrated.
  • highly reliable labeling treatment, selection treatment and base sequence determination suitable for handling a trace amount of genetic material can be performed.
  • FIG. 1 is a diagram showing a stage according to an i-th embodiment.
  • FIG. 2 is a flowchart according to the second embodiment. BEST MODE FOR CARRYING OUT THE INVENTION
  • FIG. 1 shows an entire automatic dispensing system according to the present embodiment.
  • the automatic dispensing system according to the present embodiment has a stage 10 and a dispenser (not shown).
  • the dispenser is equipped with a nozzle head provided with eight nozzles to which a detachable tip is attached, and a suction head for sucking and discharging liquid. It has a pull-discharge mechanism and eight magnetic force parts capable of applying or removing a magnetic field from outside the liquid passage of the tip mounted on the nozzle.
  • the dispenser has a moving unit that can move the nozzle head of the dispenser in parallel with the plane of the stage 10 and in the vertical direction.
  • the stage 10 includes a group of storage units for storing all substances (including magnetic particles), reagents, and liquids such as water necessary for the labeling process, and a constant temperature function for cooling or heating.
  • a group of storage units each having an unused or reusable pipe tip that can be mounted on the nozzle of the dispenser and a group of storage units that respectively store the chips for disposal detached from the dispenser.
  • a plurality of containers in which chips and racks are arranged in a matrix are prepared in advance.
  • the system has a control unit for controlling the suction and discharge of the suction and discharge mechanism, controlling the magnetic field of the magnetic force unit, and controlling the movement.
  • the control unit includes a processing unit with a built-in computer, an output unit such as a display unit such as a CR and a liquid crystal, and a printer, and a key for setting and instructing various processing procedures, inputting data, and the like. It has input means such as a reading device for reading a recording medium on which programs and data such as a board, a mouse, a floppy disk, a CD and a M ⁇ are recorded, and a communication unit connected to a communication network.
  • stage 10 in Fig. 1 there are a number of bit chips. Are arranged and held in a matrix of 8 rows x 12 columns. 'Rack 11 a, lib, 11 c, 11, and a large number of storage units (pells), each containing a cutting buffer, a synthesizing buffer, and a labeling buffer solution in each row, 4 8 Microplates (vessels) 17 arranged in rows and columns of X12 columns, ethanol tanks 12 containing ethanol, and magnetic particles containing a suspension in which 5 ⁇ particles are suspended Tank 13, water, 3 M acetic acid aqueous solution, Nut buffer solution are stored in each column, and a microplate 15 of 8 rows x 3 columns is provided, and a waste liquid tank 16 for storing waste liquid is provided. You.
  • a heating thermostat i8 for performing amplification by the PCR method and a thermostat 19 for cooling (a storage unit having a thermostat function) are provided. Have been killed. Except for the thermostats 18 and 19, the temperature was kept at room temperature (RT).
  • RT room temperature
  • the necessary bit chips, reagents, magnetic particles, samples, and the like are prepared in a storage unit or a rack in advance, so that time and effort for gene manipulation and the like are required. This process can be performed consistently and efficiently.
  • a second embodiment will be described with reference to FIG.
  • FIG. 2 shows a flowchart of the process according to the present embodiment.
  • the processing according to the present embodiment includes an extraction step (S 1 -S 2) of extracting cDNA, which is a genetic material having a specific base sequence, and a fluorescent substance, which binds to the cDNA and is a labeling substance.
  • a separation step (S8 to S1OA, S11) of adsorbing and separating on the inner wall of the substrate, and a fragmentation in which the magnetic particles are resuspended in a hydrolysis solution to generate and elute labeled RNA fragments
  • the labeled RNA fragment is obtained by repeating the steps (S 1 OB, S 1, S 13, S 14 B) and repeating the capture, separation, and elution of the eluted labeled RNA fragment with new magnetic particles at least once.
  • a purification step (S14A, S16) for purification.
  • Step S1 and step S2 are cutting steps for cutting the extracted plasmid.
  • step S1 the reusable tip rack 11 A pipe tip held in 8 rows x 1 column of 1d is attached to each of the eight nozzles of the dispenser. Then, from the 960-liter to the lm-liter of water (1) contained in the cassette-shaped container 15, each 10-liter was aspirated by the dispenser, and the heating thermostat 18 was used. Dispense into each of the 8 rows x 3 columns of storage units ([1]) in [1]. At this time, each tip of the tip is once brought into contact with the inner bottom of the empty storage part to confirm the inner bottom position, and then the liquid is discharged from the tip so that the liquid can be discharged.
  • the liquid in the pipette tip is ejected at a position slightly raised, for example, about 0.1 mm from the inner bottom so that the liquid can move to the inner bottom.
  • an extremely small amount of liquid sucked into the pipette tip is moved to the storage part by utilizing the adhesive force due to the surface tension of the inner bottom of the storage part having a larger surface area than the pipette tip. (Hereinafter referred to as “landing mode”). Since the tip is not in contact with other reagents or the like, the tip is detached and held at the position where the force of the tip was held on the reusable tip '11d. .
  • the pipe tip in the row next to the row where the detached chips are arranged in the reusable tip rack 11 d is attached to the nozzle of the dispenser, and further cut.
  • 4 liters of the buffer solution for cutting (2) is sucked from the microplate 17 containing the buffer solution for heating (2), and is pipetted into the [1] of the thermostatic bath 18 for heating. Discharge with the tip of the tip inserted into the liquid (hereinafter referred to as “in the liquid”).
  • in the liquid After detaching the tip chip from the reusable chip rack 1Id and holding it in the rack 1Id, dispensing a new tip chip from the tip rack 11c.
  • the nozzle (3) attached to each nozzle of the machine was used to suck 5 liters of the plasma (3) housed in the cooling oven 19 maintained at a temperature of 40 ° C. using the pit tip. Dispense the liquid into each of the 8 rows x 3 columns of [1] of the heating oven 18 [1].
  • the bit tip is attached to and detached from the rack 11c holding the bit tip, and is retained for disposal.
  • one unused row of tip tips is attached to each nozzle of the dispenser from the reusable rack 11 d, and is used for the cutting stored in the cooling thermostat 19.
  • the suspension in which the enzyme (4) is suspended is aspirated by 2 u liter, and discharged into a pair of cells [1] of a heating constant temperature bath 18. d It is detached and held in its original position.
  • step S2 a total of 21 liters of the suspension contained in the heating thermostat 18 is heated at 37 ° C for about 1 to 3 hours. This results in cleavage of the circular plasmid into which the cDNA having the specific nucleotide sequence has been incorporated.
  • Step S3 to Step S7 are a synthesis step of synthesizing the labeled RNA. The details will be described below.
  • step S3 after being used in step S1, the water (1) pipe tip held in the reuse rack 1 1d is attached again, and the water (1) is stored.
  • Eight liters are sucked from the cassette-shaped container 15 and discharged to the pool (housing part) group [2] of the heating thermostat 18 in the landing mode.
  • the bit tip is detached and attached to the reuse rack 11 d again.
  • An unused pipe tip held in the re-use rack 11d is attached to the nozzle of the dispenser, and a cooling thermostat containing an RN ace inhibitor (7) is attached.
  • a 2 u liter is sucked from 19 and discharged into a liquid [2] of the heating thermostat 18 in a liquid.
  • the pipe chip is an unused pipe chip from the rack 11c after being detached and held at the original position of the reuse rack 11d. Is attached, the synthesis buffer (5) contained in the microplate 17 is sucked in by 4 u liter, and is discharged in the liquid into the well [2] of the heating thermostat 18. The pit tip. Is detached at the original position of the rack 11c where the chip was held, and is discarded. An unused pipe tip held in the rack 11c was attached to the nozzle of the dispenser, and the microplate 17 containing a mixed solution of the labeled substance (6) was attached to the pipette nozzle. Aspirate the liquid by 2 liters and discharge it into the liquid [2] of the heating oven 18. The pit chip.
  • step S4 Aspirate and discharge the liquid in the liquid [2] of the heating oven 18 in step S4.
  • step S4 the heating oven 18 containing 4 liters of 20 liters obtained in this manner is placed in the heating oven 18. Heat at 37 ° C for about 1 hour to accelerate the reaction, synthesize RNA and label with fluorescent material.
  • step S5 the pipe tip mounted on the dispenser for sampling is detached and held at the original position where the pipe tip was held.
  • An unused pit tip held in the rack I1a is provided in the dispenser.
  • the reaction solution contained in the well [2] of the heating thermostat 18 is sucked in by 2 u liter, and discharged into the chiller [3] of the cooling bath 19 for reaction. Stop.
  • the reaction solution is used to confirm probe synthesis by electrophoresis (2% agarose gel).
  • step S6 the dispenser removes and holds the bit tip at the original position of the rack 11a where the tip was held, and then uses the rack 11 in step S3.
  • 2 liters of air were sucked from the cooling bath 19 containing the RN ace inhibitor (7), and the heating bath was heated.
  • 18 Discharge in liquid in well [2].
  • the unused pipette tip held in the rack 11d is mounted, and a cooling thermostat in which four RN ace polymerases (8) are stored. From 19, two liters of suction are sucked and discharged in liquid into the well [2] of the heating thermostat 18.
  • step S7 the well [2] containing a total of 22 liters of the suspension is maintained at a temperature of 37 ° C for about 1 hour for incubation.
  • a labeled RNA labeled with a fluorescent substance is synthesized.
  • Step S8 to Step S10A and Step S11 the synthesized labeled RNA is captured by magnetic particles, and the magnetic particles are adsorbed on the inner wall of the pipet chip of the dispenser.
  • This is a separation process that separates Huff.
  • Steps S12, S13 and S14B are separation steps in which the separated magnetic particles are resuspended in a hydrolyzate to purify and elute the labeled RNA fragment.
  • Step 14A and step S16 are a purification step of purifying the labeled RNA fragment by repeating capture, separation, and elution of the eluted labeled RNA fragment with new magnetic particles at least once. The details will be described below.
  • step S8 the pipette tip is detached and held at the original position of the rack 11d, and then the pipette tip used in step S6 is attached again, and the RN ace inhibitor (7) is stored.
  • a 2 u liter of the cooled thermostat 19 was sucked out and discharged in liquid into the heating thermostat 18 well [2], and the pipe tip was placed at the original position of the rack 11 d.
  • step S9 a total of 26 liters of the suspension contained in the well [2] of the heating oven 18 is heated at 37 ° C for 15 minutes.
  • step S1OA after the pipe tip is attached to and detached from the rack 11a, an unused bit chip held in the rack i1b.
  • the 3M acetic acid solution (11) contained in the force-set container 15 is sucked in by 3 liters, and discharged into the heating thermostat 18 in the liquid [2].
  • the pit tip The unused bit chips that are held in the original rack 11a and then held in the reusable rack 11d.
  • the pipette tip is not contaminated by contact with the liquid and the relatively large liquid volume of “100 w liters” allows the liquid to be transferred to the tip even if it is not settled in the bottom mode or discharged in the liquid. This is because they can be moved from the container to the storage unit, and the liquid volume does not need to be so precise.
  • the pit tip is used for reuse. 1 1d is detached and held in its original position. After attaching an unused pipe tip held in the reuse rack 11 d to the dispenser, the magnetic particle suspension contained in the magnetic particle suspension tank 13 was added to the dispenser. Absorbs 50 liters and discharges it in the air to the heating chamber 18 [2]. This is also because, as mentioned above, to prevent contamination due to contact and because it is a relatively large amount of “50 liters”, it is not necessary to perform the landing mode.
  • step S10B the pipe tip is attached and detached at the original position of the reusable rack 11d, and the unused bit tip held in the rack 11b is removed. Attached, 200 ⁇ l of the alkaline hydrolyzed solution contained in the alkaline hydro tank 14 is suctioned and discharged into the heating thermostat tank 18 [4].
  • step S11 the pipette tip of the dispenser is attached to and detached from the reuse rack 11d to its original position, and then the unused pipe tips held in the rack 11b are removed. A pet tip was attached, and the liquid of the pipe tip was applied to the suspension of 179 u liters contained in the heating thermostat i8 using magnetic means provided in the dispenser. When suction and discharge are performed in a state where a magnetic field is applied in the passage, or by repeating suction and discharge, the magnetic particles are adsorbed and separated on the inner wall of the pipe tip.
  • step S12 the magnetic particles are transferred to the container [4] containing the ethanol (12) of the thermostat 18 for caro heating while being held by the piget tip, and the magnetic force means applies a magnetic field.
  • the magnetic particles are resuspended in ethanol (12) by repeatedly sucking and discharging the ethanol under the condition that the magnetic particles are not affected.
  • the magnetic particles are resuspended in ethanol (12), and DNA having a predetermined base sequence, which is the target substance, is eluted from the magnetic particles.
  • the dispenser repeats suction and discharge of the liquid while the magnetic force exerts a magnetic field inside the bit tip, so that only the magnetic particles are deposited on the inner wall of the bit tip.
  • the magnetic particles are separated and transferred to a waste liquid tank 16 while adsorbing the magnetic particles, and the magnetic particles are discarded by suction and discharge in a state where the magnetic force means does not apply a magnetic field.
  • step S 13 after that, it was stored in the well [4] of the heating oven 18.
  • the eluate was incubated at a temperature of about 60 ° C for about 10-60 minutes.
  • step S14A the dispenser attaches and detaches the pipet tip to and from the original position of the rack 11b, and then repeats step S held in the reusable rack 11d. Re-attach the bit tip used in 3 etc. again, aspirate 20 liters of water (1) contained in the cassette 15 and discharge it to the microwell 17 [5] .
  • step S14B with the pipet tip attached, 20 liters of suction from the cassette-like container 15 containing the buffer solution for neutralization was sucked, and the well of the thermostatic bath 18 for heating [4] Then, eject the tip in the air without immersing the tip of the tip in the reaction solution.
  • the pit chip Is attached to and detached from the reusable rack 11d.
  • a pipe tip held in the reusable rack 11 d is mounted, and the magnetic particles are suspended from a magnetic particle suspension tank 13 containing a suspension in which the magnetic particles are suspended.
  • the suspension is suctioned at 50 liters and discharged into the heating thermostat 18 in the air.
  • the pipe tip is detached from the reuse rack 11 d and held.
  • the pit chip used in step S10 held in the reuse rack 11d. Is re-attached, and the ethanol solution contained in the ethanol tank 12 is suctioned at 400 uL and discharged into the heating thermostat tank 18 [4] in the air.
  • the pit tip Is detached and held by the reusable rack 11d in which the bit tip was held.
  • Step S15 the suspension (4) of the heating thermostatic bath 18 containing a total amount of 670 uL was heated at 60 ° C and held in the rack 11b.
  • the pipet tip is mounted on the nozzle of the dispenser, and the suction tip I and the discharge are repeated in a state where a magnetic field is applied to the inside of the pipet tip by a magnetic force means.
  • the magnetic particles are separated by being adsorbed on the inner wall of the substrate.
  • step S16 with the magnetic field applied by the magnetic means, the magnetic particles are transferred to the microplate 17 with the magnetic particles kept separated, and the magnetic field by the magnetic means is removed.
  • the water contained in the well [5] is repeatedly suspended and dissolved in the water by repeating suction and discharge.
  • a magnetic field is applied to the inside of the pipe tip by magnetic force means, and only the magnetic particles from which the target substance has separated are separated on the inner wall of the pipe tip.
  • the chip is transported to the waste tank 16 and the magnetic particles are discarded in the waste tank 16 by repeating suction and discharge without applying a magnetic field by magnetic means.
  • the automatic target substance sorting method is performed using the dispenser according to the first embodiment.
  • a chip rack in which a large number of bit chips are arranged, a tank containing pure water, a container containing a suspension in which cells and tissues are suspended, an oligo dT A tank for holding a suspension in which the first magnetic particles coated with, and a suspension in which the second magnetic particles coated with one of the specific binding compound pairs are suspended.
  • An accommodating section or the like for accommodating is prepared on the stage.
  • an extraction step (S21) of extracting RNA, which is a genetic material, from cells or tissues, and cDN, a related genetic material of the RNA, using a reverse transcriptase is a method of extracting RNA, which is a genetic material, from cells or tissues, and cDN, a related genetic material of the RNA, using a reverse transcriptase.
  • A and a cDNA synthesis step (S22) for synthesizing cDNA fragments, which are various types of related genetic material fragments, and a PCR primer that binds to the cDNA fragment via an adapter
  • a piotinylated RNA fragment labeled with piotin and a second magnetic particle coated with streptavidin the other side of the compound pair.
  • RNA is extracted from cells and tissues.
  • SDS and protease the necessary reagents
  • the first magnetic particles are applied to the suspension of the sample in which the protein or the like is dissolved, and the suspension is accommodated in a state where a magnetic field is applied to the inner wall of the pipe tip by the magnetic force means.
  • the sample is sucked from the container, adsorbed, moved, and discharged into the sample suspension to be mixed.
  • the genetic material such as RNA is captured by the first magnetic particles.
  • a magnet M provided as a magnetic force outside the pipe tip, for example, a magnet M which can be freely attached and detached is provided on the outer peripheral surface of the pipette tip.
  • the first magnetic particles on which RNA and the like are captured are made to adhere to the inner surface of the pit chip.
  • an ethanol solution was added to the solution, and the suction and discharge were repeated to precipitate in the ethanol solution.
  • step S22 the pit tip is mounted from the tip rack, and a reverse transcriptase suspension is sucked from a storage section in which reverse transcriptase is stored, and only the RNA and the like are suspended.
  • a cDNA fragment complementary to the RNA is synthesized in a state of forming a double strand with the RNA fragment.
  • the RNA degrading enzyme is discharged into the suspension to obtain a single-chain cDNA.
  • step S23 the primer for PCR is bound to the cDNA via the T4RNA ligase (ligase) which is an adapter to the single-stranded cDNA fragment. Thereafter, the cDNA is amplified by a PCR method.
  • T4RNA ligase ligase
  • step S24 the amplified cDNA fragments and the specific nucleotide sequence A biotinylated RNA having a specific base sequence labeled with biotin, which is one of a pair of compounds that specifically binds (using the labeled RNA obtained according to the second embodiment).
  • a biotinylated RNA having a specific base sequence labeled with biotin which is one of a pair of compounds that specifically binds (using the labeled RNA obtained according to the second embodiment).
  • Is aspirated from the container containing the liquid in which is suspended discharged into the container containing the cDNA suspension, and mixed.
  • the above-mentioned dispenser discharges the second magnetic particles into the storage portion storing the suspension, thereby capturing the force of the second magnetic particles.
  • the dispenser repeats the suction and discharge of the suspension while attaching a new bit tip and then applying a magnetic field to the liquid passage of the bit tip by magnetic means.
  • the second magnetic particles are adsorbed on and separated from the inner wall of the pipe tip. After the residual liquid is discarded, the liquid is resuspended by repeatedly adding and discharging the ethanol liquid without applying the magnetic field by the magnetic force means, and the second magnetic particles are precipitated in the ethanol liquid. Let it. Thereafter, the eluate is added to elute the cDNA fragment as the target substance from the second magnetic particles.
  • the suspension is repeatedly suctioned and discharged while a magnetic field is applied to the pipet tip of the pipettor by a magnetic force means, so that only the second magnetic particles from which the cDNA fragments have been eluted are pipetted.
  • a magnetic force means By adsorbing on the inner wall of the chip and separating and discarding, the target cDNA is selected.
  • the method according to the fourth embodiment uses a dispenser and a DNA chip, which have already been described, to determine a base sequence. Is what you do.
  • the method comprises an extraction step (S31) of extracting RNA, which is a genetic material, from a sample such as a cell or a tissue, and a fluorescently labeled cDNA that is complementary to the RNA as a labeling-related genetic material.
  • the labeled cDNA is captured by magnetic particles by sucking each liquid from the storage part and moving and discharging the liquid into a thermostat, and the suspension passes through the liquid passage.
  • a purification step (S33) in which a magnetic field is applied during the passage to perform adsorption and separation on the inner surface of the liquid passage and elution is performed several times, and the labeled cDNA suspension is aspirated.
  • step S31 The extraction process in step S31 is the same as the process described in the third embodiment, and thus the description is omitted.
  • step S32 is the same as the synthesizing step described in the step S22 of the third embodiment, and the description is omitted.
  • step of purifying step S33 in order to capture the labeled cDNA, the suspension is performed by mixing the magnetic particles and the ethanol coated with oligo dT that specifically binds to the cDNA and the suspension. To allow the magnetic particles to capture the labeled cDNA. Next, a magnetic field is applied to the inside of the tip by the magnetic means of the dispenser, and the suction and discharge of the suspension are repeated to adsorb and separate on the inner wall of the tip. I do.
  • the residual liquid is discarded, and the target cDNA is eluted from the magnetic particles with the pure water or the like. This separation process is repeated several times to purify the target substance, labeled cDNA.
  • the labeled cDNA purified in step S34 is discharged onto a DNA chip, and the base sequence of the cDNA is determined.
  • the transfer of a liquid or the like is performed by moving the dispenser, and the transfer of the liquid or the like is performed by moving the force, the stage or the container on the stage as described above.
  • the container and the dispenser may be movable.
  • the case of synthesizing cDNA using reverse transcriptase has been described.However, the DNA is amplified by PCR using the primary, and the amplified DNA is placed on a DNA chip. What discharges may be sufficient. Also, it is needless to say that the numerical values used are merely examples and are not limited to these numerical values.
  • the dispensing system is used in the case of eight nozzles
  • the case where a disposable tip is used has been described.
  • the number of a plurality of sets is not limited to this case, and a case where a cleaning nozzle is used instead of the disposable tip may be used.

Abstract

L'invention concerne un procédé d'étiquetage automatique faisant appel à un distributeur automatique très fiable qui effectue automatiquement un traitement très efficace avec une charge allégée sur l'opérateur sans augmenter les dimensions du dispositif et convenant à la manipulation d'une substance génétique, un procédé de séparation automatique d'une substance cible, un procédé permettant de déterminer une séquence de base et un système de distribution automatique.
PCT/JP2000/005305 1999-08-09 2000-08-08 Procede d'etiquetage automatique faisant appel a un distributeur automatique, procede de separation automatique d'une substance cible, procede permettant de determiner la sequence de base et systeme de distribution automatique WO2001011364A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP22566899 1999-08-09
JP11/225668 1999-08-09

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WO2001011364A1 true WO2001011364A1 (fr) 2001-02-15

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Publication number Priority date Publication date Assignee Title
EP2056303A1 (fr) * 2007-10-29 2009-05-06 Atomic Energy Council - Institute of Nuclear Energy Research Système de délivrance d'isotopes liquides
JP2010127941A (ja) * 2008-11-28 2010-06-10 F Hoffmann-La Roche Ag 核酸の自動抽出システムおよびその方法
WO2010143295A1 (fr) * 2009-06-12 2010-12-16 株式会社島津製作所 Procédé d'amplification de gènes à multiples étapes
JP2012013697A (ja) * 2010-06-29 2012-01-19 F. Hoffmann-La Roche Ag サンプルの分配
WO2014189085A1 (fr) * 2013-05-21 2014-11-27 ユニバーサル・バイオ・リサーチ株式会社 Dispositif et procédé de prétraitement de séquenceur
JP2017072603A (ja) * 2008-12-25 2017-04-13 ユニバーサル・バイオ・リサーチ株式会社 生体関連物質測定装置
JP2020537143A (ja) * 2017-10-11 2020-12-17 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 磁気粒子を用いて生物学的試料をプロセシングするための方法

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JPH04501956A (ja) * 1988-11-21 1992-04-09 ダイナル・エイ・エス Ｒｎａ及びｄｎａの検出並びに定量法
WO1996041811A1 (fr) * 1995-06-08 1996-12-27 Boehringer Mannheim Gmbh Pigment magnetique
JPH0919292A (ja) * 1995-07-07 1997-01-21 Toyobo Co Ltd 核酸結合用磁性担体およびそれを用いる核酸単離方法
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JPH01201156A (ja) * 1987-11-16 1989-08-14 Gene Trak Syst 磁気的分離デバイスおよび不均質検定における使用法
JPH04501956A (ja) * 1988-11-21 1992-04-09 ダイナル・エイ・エス Ｒｎａ及びｄｎａの検出並びに定量法
WO1996041811A1 (fr) * 1995-06-08 1996-12-27 Boehringer Mannheim Gmbh Pigment magnetique
JPH0919292A (ja) * 1995-07-07 1997-01-21 Toyobo Co Ltd 核酸結合用磁性担体およびそれを用いる核酸単離方法
JPH09218201A (ja) * 1995-12-07 1997-08-19 Seiko Instr Inc 磁性粒子の分離方法

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2056303A1 (fr) * 2007-10-29 2009-05-06 Atomic Energy Council - Institute of Nuclear Energy Research Système de délivrance d'isotopes liquides
JP2010127941A (ja) * 2008-11-28 2010-06-10 F Hoffmann-La Roche Ag 核酸の自動抽出システムおよびその方法
JP2017072603A (ja) * 2008-12-25 2017-04-13 ユニバーサル・バイオ・リサーチ株式会社 生体関連物質測定装置
WO2010143295A1 (fr) * 2009-06-12 2010-12-16 株式会社島津製作所 Procédé d'amplification de gènes à multiples étapes
JP2012013697A (ja) * 2010-06-29 2012-01-19 F. Hoffmann-La Roche Ag サンプルの分配
WO2014189085A1 (fr) * 2013-05-21 2014-11-27 ユニバーサル・バイオ・リサーチ株式会社 Dispositif et procédé de prétraitement de séquenceur
JPWO2014189085A1 (ja) * 2013-05-21 2017-02-23 ユニバーサル・バイオ・リサーチ株式会社 シーケンサ前処理装置およびその方法
US10626440B2 (en) 2013-05-21 2020-04-21 Universal Bio Research Co., Ltd. Sequencer pretreatment device and method thereof
JP2020537143A (ja) * 2017-10-11 2020-12-17 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 磁気粒子を用いて生物学的試料をプロセシングするための方法
JP7155258B2 (ja) 2017-10-11 2022-10-18 エフ．ホフマン－ラ ロシュ アーゲー 磁気粒子を用いて生物学的試料をプロセシングするための方法

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