JP2825008B2 - Nucleic acid sample preparation equipment - Google Patents

Description

The present invention relates to an apparatus for preparing a sample of a biological substance such as a nucleic acid.

[Conventional technology]

DNA sequencing is a fundamental technology in life science. However, the sample preparation process is complicated and burdensome for researchers, and skill is required to stably prepare DNA samples. For example, it takes about 5 days at the earliest to determine the DNA base sequence of 1 kbp, but the actual working time of the operator in the sample preparation process is nearly 20 hours, which is a heavy burden. In order to accelerate life science research in the future, it is essential that anyone can easily and reliably prepare DNA samples.

Conventionally, an automatic device for the complementary strand synthesis reaction in the DNA sample preparation process (BIO / TECHNOLOGY) 6
Volume 1988 October pp. 1211-1213) was developed. This automatic device consists of a dispensing mechanism that aspirates and discharges a liquid and shifts the phase, and a sample tray that has a large number of cavities for mixing the liquid.
Labor saving of about 5 to 10% of the sample preparation process can be achieved.

[Problems to be solved by the invention]

However, the DNA sample preparation process often involves alternating enzyme reactions and purification operations, and in order to expand the automation range and further improve the labor saving effect, the enzyme reactions and purification operations can be performed sequentially with a single device. Automation is essential. In order to perform DNA purification and reaction sequentially, liquid loss and cross-contamination due to splashing and evaporation of liquid can be prevented, and a lid that can be fitted is integrally molded and withstands centrifugal acceleration of 10,000 G or more. A mechanism capable of carrying, dispensing, opening and closing, dispensing, centrifuging, mixing, and drying of a disposable container is required, but in the above automatic device having a dispensing mechanism and a cavity for mixing a liquid as basic elements, The purification operation cannot be automated.

The present invention has been made in order to solve the above-mentioned problems, and has as its object to provide a sample preparation apparatus capable of sequentially performing an enzyme reaction and a purification operation.

[Means for solving the problem]

A container made by integrally molding a lid that can be fitted,
A lid opening mechanism for opening the lid of the container, a lid closing mechanism for closing the lid of the container, a dispenser for dispensing liquid from the container to another container after opening the lid of the container, and a predetermined dispenser for the container. One or both dispensers for dispensing a liquid sample from a sample bottle, and the lid opening mechanism,
The lid closing mechanism, a transport device that transports the container to the dispenser, a centrifuge that applies a centrifugal acceleration of 10,000 G or more to separate the liquid in the container, and a mixer that mixes the liquid in the container And a cold storage room for storing the liquid in the container,
An incubator that heats the liquid in the container, a container reversing device that discards the liquid in the container, a dryer that dries the liquid in the container, the container, the transfer device, the centrifuge, The mixer, the cool room, the incubator, a transporter for transporting between the dryers, the lid opening mechanism, the lid closing mechanism, the dispenser, the transporter, the centrifuge, the mixer, the transporter A controller for controlling a cool room, the incubator, the container reversing machine, the drying machine, and the transporting machine, wherein the lid opening mechanism, the lid closing mechanism, the dispensing machine, the transporting apparatus, and the centrifugal separator are provided. The transporter is sequentially operated according to the process data stored in the controller, and the mixing, reaction, extraction, and purification of the nucleic acid sample and the sample are performed fully automatically.

Further, a container made by integrally molding a fittable lid, a lid opening mechanism for opening the lid of the container, a lid closing mechanism for closing the lid of the container, and a container after opening the lid of the container. One or both of a dispenser for dispensing a liquid into a separate container and a dispenser for dispensing a liquid sample from a predetermined sample bottle to the container, the lid opening mechanism, and the lid closing mechanism A transport device that transports the container to the dispenser, a centrifugal separator that applies a centrifugal acceleration of 10,000 G or more to separate the liquid in the container, a mixer that mixes the liquid in the container, and the container A cold storage room for storing the liquid in the container, and an incubator for heating the liquid in the container,
A container reversing device for discarding the liquid in the container, a dryer for chaptering the liquid in the container, and the container, the transfer device, the centrifugal separator, the mixer, the cool room, the incubator, A transporter for transporting between dryers, the lid opening mechanism, the lid closing mechanism, the dispenser, the transporting device, the centrifuge, the mixer, the cool room, the incubator, the container reversing machine, The dryer, comprising a controller for controlling the transporter, a predetermined sample and a predetermined buffer solution held in a container in the cold room, a plurality of reagents containing enzymes, the transported by the transporter by the transporter Transport to the device, the transport device, the lid opening mechanism,
The dispenser, the lid of the container is opened by a predetermined operation of a lid closing mechanism, the sample and the reagent are dispensed and mixed, the lid is closed, and the container holding the dispensed and mixed liquid is transferred to the transporter. The first step consisting of transporting and mixing the mixed liquid and transporting the container holding the mixed liquid to the incubator by the transporter and heating the sample liquid after the first step, Conveyed to the transport device by a transport device, the transport device, the lid opening mechanism, the dispenser, the lid of the container is opened by a predetermined operation of the lid closing mechanism, the sample liquid and a predetermined salt, The phenol and chloroform aqueous solution reagents are dispensed and mixed, the lid is closed, the container holding the dispensed and mixed liquid is transported to the mixer by the transporter and mixed, and the mixed liquid is mixed. The transported container is transported to the centrifugal separator by the transporter and centrifuged, and the container holding the separated liquid is transported to the transporter by the transporter, the lid is opened by the lid opening mechanism, and the dispensing is performed. Transfer the supernatant of the separated liquid to a new container by a machine, the second step consisting of closing the lid of the container holding the transferred liquid by the lid closing mechanism, the first step or The container holding the sample liquid after the second process is transferred to the transfer device by the transfer device, and the transfer device, the lid opening mechanism, the dispenser, and the lid closing mechanism are operated by a predetermined operation. Open the lid of the container, dispense and mix the sample liquid, the predetermined salt, and the ethanol reagent, close the lid, and transfer the container holding the dispensed and mixed liquid to the mixer by the transfer device. Then mix The container holding the mixed solution is transferred to the centrifugal separator by the transfer device and centrifuged, and the container holding the separated liquid is transferred to the transfer device by the transfer device and the lid opening mechanism is used. Open the lid, discard the supernatant of the separated solution by the dispenser or the container reversing machine, transport the container holding the sample after the supernatant was discarded to the dryer by the transporter, Drying the liquid remaining after disposal, transporting the dried container to the transport device by the transporter, dispensing a predetermined liquid, and closing the lid by the lid closing mechanism, a third process, Preparation of a nucleic acid sample consisting of one step or a combination of a plurality of steps is performed fully automatically and sequentially.

[Action]

This nucleic acid sample preparation device is provided with sufficient and essential device elements for nucleic acid sample preparation.

〔Example〕

FIG. 1 is a schematic perspective view showing a sample preparation device according to the present invention, FIG. 2 is a plan view showing a transfer device of the dispenser, FIG. 3 is a diagram showing a lid opening mechanism of the dispenser, FIG. Is a diagram showing a lid closing mechanism of the dispenser, FIG. 5 is a diagram showing a transfer dispenser, FIG. 6 is a diagram showing a configuration of a pressure dispenser, FIG. 7 is a plan view showing a centrifuge, 8 is a sectional view showing a centrifugal bucket of a centrifuge, FIG. 9 is a view showing a mixer, FIG. 10 is a perspective view showing an incubator, FIG. 11 is a view showing a container reversing machine, and FIG. It is a figure showing a grasping mechanism part of a conveying machine.

In the figure, 1 is a container, 1a is a lid which can be fitted to the container 1, and the container 1 and the lid 1a are made by integral molding,
The container 1 is made of a lightweight and high-strength plastic such as polypropylene, and the container 1 can withstand a centrifugal acceleration of 10,000 G or more. 17 is a machine room.

Reference numeral 7 denotes a dispenser for dispensing a liquid such as a reagent into the container 1, and reference numeral 2 denotes a transfer device of the dispenser 7, and the transfer device 2 transfers the container 1 on a plane. 2a and 2b are turntables of the transfer device 2, 18a and 1
8b is a pulse motor for driving the turntables 2a and 2b, 19
a and 19b are belts hung between the output shafts of the pulse motors 18a and 18b and the turntables 2a and 2b, and 20 is a turntable 2
a, a container mounting hole provided in 2b, 24 is a lever provided in the container mounting hole 20, 23 is an elastic member pressing the lever 24, 3 is a lid 1a of the container 1 conveyed by the turntables 2a, 2b.
, An opening mechanism 29, a holding table fixed to the machine room 17, a gear 28 attached to the holding table 29, and the gear 28 driven by a motor (not shown). 27a and 27b are gears attached to the holding base 29, and the gears 27a and 27b mesh with the gear. Reference numeral 25 denotes a hook member rotatably attached to the gears 27a and 27b by pins 26a and 26b. Reference numeral 25a denotes a tip of the hook member 25, and the tip 25a is shaped to hook the lid 1a. 4 is a lid closing mechanism for closing the lid 1a of the container 1 transported by the turntables 2a and 2b, and 35 is a machine room
A holding base fixed to 17 is a gear attached to the holding base 35, and the gear 34 is driven by a motor (not shown). 33a and 33b are gears attached to the holding base 35,
33a and 33b are in mesh with the gear 34. 31 is pins 32a, 32b
A lid push-down member rotatably attached to the gears 33a and 33b by the lever 31a is a tip provided on the lid push-down member 31, and 31b is a roller provided on the tip 31a. 5
Is a disposable chip, 6 is a chip supply mechanism for supplying the disposable chip 5, 6a is a storage section for storing the disposable chip 5, 6b is a transport mechanism for transporting the storage section 6a, and chip supply is performed by the storage section 6a and the transport mechanism 6b. The mechanism 6 is constituted. Reference numeral 15 denotes a chip disposal hole provided between the transfer device 2 and the chip supply device 6. 22 is a transfer dispenser of the dispenser 7, and 52 is a machine room 17
, A moving member movably attached to the holding table 52, 50 is a holding plate fixed to the moving member 51, and the moving member 51 and the holding plate 50 are driven by a belt, a motor, and the like. It is moved left and right in FIG. 5 by an apparatus (not shown). 49 is a pulse motor attached to the holding plate 50, 47b is a male screw rotatably attached to the holding plate 50, 48 is a coupling connecting the output shaft of the pulse motor 49 and the male screw 47b, 46 is a holding plate A holding plate movably attached to 50, 47a is a female screw fixed to the holding plate 46, a female screw 47a is screwed into a male screw 47b, and the holding plate 50 has an origin of the vertical movement operation, A position sensor (not shown) for specifying the upper and lower limits is provided. 37a and 37b are holding plates
The body portions 37a and 37b attached to 46 include a plunger (not shown), and the diameter of the plunger included in the body portion 37a is smaller than the diameter of the plunger included in the body portion 37b. 38a, 38b are disposable tip mounting portions provided at the tips of the body portions 37a, 37b, 44 is a pulse motor mounted on the holding plate 46, 42b is a male screw rotatably mounted on the holding plate 46, 43 is a pulse A coupling for connecting the output shaft of the motor 44 and the male screw 42b, 42a is a female screw screwed to the male screw 42b, and a plunger is attached to the female screw 42a. 39a, 39b are covers attached to female screw 42a, 40
a and 40b are chip removing members for removing the disposable chip 5 attached to the chip attaching portion 38 by being pushed by the covers 39a and 39b, 41 is a cover holding member for holding the covers 39a and 39b, and 45 is provided on a holding plate 46. The position sensor 45 designates the origin and upper and lower limits of the covers 39a and 39b. Then, the chip supply mechanism 6, the chip disposal hole 15, the holding table 52, the holding plate 50, the pulse motor 49,
The transfer dispenser 22 is constituted by the holding plate 46, the pulse motor 44, and the like. 21 is a pressure-feeding dispenser, 8 is a closed container, 8a is a liquid contained in the closed container 8, 21c is a solenoid valve connected to the closed container 8 by an air pipe 21b, 21e is an air tank connected to the solenoid valve 21c. , 21f is a pressure regulating valve connected to the air tank 21c, 21i is a high-pressure air source connected to the air regulating valve 21f, 21
d is an electromagnetic valve connected to the electromagnetic valve 21c, 21g is an atmospheric pressure source 21h connected to the electromagnetic valve 21d, a vacuum pump connected to the electromagnetic valve 21d, 21a is one end immersed in the liquid 8a, and others. The liquid pipe whose end is located above the turntable 2b is a pressure pipetting / dispensing machine 21 composed of the sealed container 8, the solenoid valves 21c and 21d, and the like. The transfer dispenser 22 and the pressure dispenser 21 constitute the dispenser 7.

In the dispenser 7, when the pulse motors 18a and 18b are operated, the turntables 2a and 2b are rotated clockwise in FIG. 2, so that the container 1 placed in the container mounting hole 20 is flattened. Can be transported up. When the gear 28 is driven clockwise by a motor, the gears 27a, 27b
3 rotates counterclockwise in FIG. 3, so that the tip portion 25a moves upward, so that the lid 1a of the container 1 can be opened. Further, if the gear 34 is driven clockwise by a motor, the gears 33a and 33b rotate counterclockwise in FIG. 4, so that the roller 31b moves in an arc shape. Can be closed. Further, if the holding plate 50 is moved by the driving device, the positions of the body portions 37a and 37b in the left-right direction in FIG. 5 can be determined. Further, by operating the pulse motor 49, the positions of the body portions 37a and 37b in the vertical direction can be determined. By operating the pulse motor 44, the vertical positions of the plunger and the covers 39a and 39b can be determined. Therefore, the tip supply mechanism 6 is driven to move the disposable chip 5 to the supply position, and the body portions 37a and 37b are positioned above the chip supply mechanism 6, and then the body portions 37a and 37b are lowered. The disposable tip 5 can be attached to the disposable tip attaching portions 38a, 38b provided at the tips of the portions 37a, 37b, and thereafter the body portion
37a, 37b are raised and moved horizontally, and the body portions 37a, 37b are positioned above the container mounting hole 20b at the dispensing position of the turntable 2a, and the body portions 37a, 37b are lowered to disposable chips. 5 is immersed in the liquid in the container 1 placed in the container mounting hole 20b, the plunger is raised to suck the liquid into the disposable tip 5, and the body portions 37a and 37b are raised and moved horizontally. Then, the body portions 37a, 37b are positioned above the container mounting hole 20c at the dispensing position of the turntable 2b, and the body portions 37a, 37b
To insert the tip of the disposable tip 5 into the container 1 placed in the container mounting hole 20c, and lower the plunger to discharge the liquid in the disposable tip 5;
The liquid can be dispensed from the container 1 placed in the container 1 into the container 1 placed in the container placing hole 20c.
7b, move horizontally to position the body portions 37a, 37b above the chip disposal hole 15, and lower the covers 39a, 39b.
The disposable tip 5 is removed from the tip mounting portions 38a and 38b by pushing the a and 40b, and the disposable tip 5 is discarded into the tip discarding hole 15. In this case, the disposable tip 5 is attached to the tip attaching portion 38a when the dispensing amount in the transfer dispenser 22 is small, and when the dispensing amount is large, the tip attaching portion 38
The disposable tip 5 is attached to b to perform the liquid suction and discharge operations. Further, the state in which the sealed container 8 and the atmospheric pressure source 21g are connected by the electromagnetic valves 21c and 21d,
Is switched to connect the closed container 8 and the high pressure air source 21i, the liquid 8a is transferred from the closed container 8 via the liquid pipe 21a to the container 1.
Supplied within. From this state, after switching the solenoid valves 21c and 21d to connect the sealed container 8 and the vacuum pump 21h,
By switching the electromagnetic valve 21d to connect the closed container 8 and the atmospheric pressure source 21g, the liquid 8a in the liquid pipe 21a can be completely drawn back into the container 1.

As described above, the lid opening mechanism 3, the dispensing position of the transfer dispenser 22, the dispensing position of the pressure dispenser 21 and the lid closing mechanism 4 are provided along the conveying device 2 in accordance with the handling operation order. The operation, the dispensing operation, and the lid closing operation can be simultaneously and efficiently performed for a plurality of containers 1. When one dispensing operation is completed, the disposable tip 5 is inserted into the tip disposal hole 15.
Because it is thrown away, contamination can be prevented. Furthermore, the transfer dispenser 22 is used for dispensing a small amount of liquid of the order of several μl with high precision, for example, for preparing an enzyme reaction solution of DNA, for collecting an aqueous layer containing DNA by phenol extraction, and for pH and salt concentration. This is effective when preparing a reaction solution that needs to be strictly maintained under predetermined conditions.On the other hand, the pumping dispenser 21 can inject a large amount of liquid in a short time. For example, when purifying using the above method or dissolving a dried DNA sample in TE buffer, the dispensing volume is as large as several tens μl and the dispensing accuracy is ±
If it is within 10%, it is suitable for the dispensing operation when reproducibility can be guaranteed. If the transfer dispensing machine 22 and the pressure dispensing dispenser 21 are provided in parallel, in the case of dispensing sodium acetate on the order of several μl and ethanol of 100 μl or more into the DNA solution in ethanol precipitation, the sodium acetate is transferred and dispensed. In addition to dispensing at 22, ethanol can be dispensed at the pressure-feeding dispenser 21, so that the processing time can be reduced. Also, unlike the dispenser using the ironing pump, the pressure-feeding dispenser 21 has no liquid at the tip of the liquid pipe 21a during non-dispensing, so that there is no danger of dropping by mistake and reliability of sample preparation. The nature also increases.

Reference numeral 11 denotes a centrifugal separator that separates the liquid in the container 1 by applying centrifugal acceleration, 68 denotes the main body of the centrifuge 11, 56 denotes a shaft directly connected to a high-speed rotation motor (not shown), and 57 denotes a shaft. 58, an outer wall of the rotor 57, 69 a steel wire attached to the rotor 57, 62 a swing rotor type centrifugal bucket supported by the steel wire 69, 59 a container insertion hole provided in the centrifugal bucket 62. , 60 are grooves provided in the centrifugal bucket 62, and the upper portion of the container 1 is engaged with the grooves 60. 61 is rotor 57
A gap adjusting plate provided between the centrifugal bucket 62 and the centrifugal bucket 62, 64 is a cam mechanism rotatably attached to the main body 68, 64a is a cam surface provided on the cam mechanism 64, and 63 is for rotating the cam mechanism 64. An air cylinder 66 is a lever rotatably provided on the main body 68, 65a to 65c are rollers attached to the lever 66, and the rollers 65a to 65c are pressed against the cam surface 64a by an elastic member (not shown). ing. Reference numeral 67 denotes a pulse motor for rotating the roller 65a, which is provided with a photo sensor (not shown) for detecting the position of the rotor 57.
4. A positioning mechanism is constituted by the lever 66, the rollers 65a to 65c, the pulse motor 67 and the like.

In this centrifuge 11, the container 1 is inserted into the container insertion hole 59.
Is inserted, the upper protrusion of the container 1 is engaged with the groove 60, and the high-speed rotation motor is operated. When the centrifugal acceleration applied to the container 1 becomes 10,000 G or more, the rotor 57 rotates at a high speed, and the centrifugal bucket 62 Can be rotated counterclockwise around the steel wire 69 in FIG. 8 to separate the liquid in the container 1 inserted into the container insertion hole 59. Then, if the air cylinder 63 is reduced after the high-speed rotation motor is stopped, the cam mechanism 64 rotates, and the rollers 65a to 65c are pushed by the cam surface 64a, so that the rollers 65a to 65c come into contact with the outer wall 58. In this state, if the pulse motor 67 is operated according to the output of the photosensor, the centrifugal bucket 62 can be brought into a state in which the container 1 can be carried in and out. Thereafter, when the air cylinder 63 is extended, the cam mechanism 64 rotates, and the rollers 65a to 65c are pressed against the cam surface 64a by the elastic member, so that the state shown in FIG. 7 is obtained.

As described above, since the groove 60 is provided in the centrifugal bucket 62, if the upper protruding portion of the container 1 is engaged with the groove 60, the container 1 can be rotated even at a high speed.
Direction can be fixed, and the groove 60 is
Since the operation space (described later) is used, the operation of grasping the container 1 can be easily performed. Further, since the rotor 57 rotates at a high speed at which the centrifugal acceleration applied to the container 1 becomes 10,000 G or more, as is clear from FIG. 13, sodium acetate and ethanol or ammonium acetate and ethanol are dispensed and mixed into the DNA solution. It is possible to maximize the amount of DNA recovered in the ethanol precipitation operation by centrifugation. Further, since the centrifugal bucket 62 rotates around the steel wire 69 during high-speed rotation, when the container 1 is carried in and out of the centrifugal bucket 62, the container insertion hole 59 is oriented in the vertical direction.
The loading / unloading operation of the container 1 can be easily performed. Therefore, in the phenol extraction used for the DNA purification operation, the container 1 is dispensed without disturbing the interface between the aqueous layer containing DNA and the phenol layer after dispensing, mixing and centrifuging phenol into the DNA solution. Can be transported.

12 is a mixer for mixing the liquid in the container 1, 70 is the mixer main body, 74 is the vortex mixer, and 72a to 72c are the mixer main body 70.
The gears 72a to 72c mesh with each other. 72d is a lever fixed to the gear 72a, 71 is the mixer body
The other end of the air cylinder 71 is pin-connected to a lever 72d. 73 is a container holding mechanism fixed to the gear 72c, 76 is a container direction correcting mechanism that corrects the direction of the container 1 by pressing a protruding portion of the container 1, and 75 is an air cylinder that drives the container direction correcting mechanism 76. At 75, the mixer 12 is constituted by the vortex mixer 74, the container holding mechanism 73, the container direction correcting mechanism 76, and the like.

In this mixer 12, after attaching the container 1,
After activating the air cylinder 71 and pressing the container 1 by the container pressing mechanism 73 and then operating the vortex mixer 74, the liquid in the container 1 can be mixed. Next,
By operating the air cylinder 75 and correcting the direction of the container 1 by the container direction correcting mechanism 76, the container 1 can be brought out.

9 is a cold storage room for storing the liquid in the container 1, 9a is a refrigerated storage room, 9b is a frozen storage room, and the upper surface of the refrigerated storage room 9a and the frozen storage room 9b is provided with a groove with which the upper part of the container 1 is engaged. Has been
The cold storage room 9 is composed of the refrigerated storage room 9a and the frozen storage room 9b.

In this cold storage room 9, if the container 1 is held in the refrigerated storage room 9a, the liquid in the container 1 can be refrigerated and stored,
If the container 1 is held in the freezing storage room 9b, the liquid in the container 1 can be stored frozen.

10 is an incubator for heating the liquid in the container 1, 87 is a closed container, 91 is the upper surface of the closed container 87, 88 is a pipe for vacuum suction connected to the closed container 87, and a pipe 88 for vacuum suction is a vacuum pump ( (Not shown).
Air suction and exhaust means for sucking and exhausting the air inside the sealed container 87 is constituted by, for example, this. 89 is an air supply pipe connected to the sealed container 87, 84 is a metal block provided in the sealed container 87, and the metal block 84 is heated by a heater (not shown). 83 is a container insertion hole provided in the metal block 84, and a groove with which the upper part of the container 1 is engaged is provided on the upper surface of the metal block 84. 86 is a shutoff lid rotatably attached to the closed container 87, 85 is an air cylinder that opens and closes the shutoff lid 86, 90 is an air cylinder that moves the closed container 87 up and down,
The incubator 10 is configured by the closed container 87, the metal block 84, the shutoff lid 86, and the like.

In this incubator 10, the container 1 is inserted into the container insertion hole 83, the air cylinder 85 is operated to close the shutoff lid 86, and the air cylinder 90 is operated to raise the closed container 87 so that the upper lid 91 and the upper lid 91 are closed. When the heater is operated after contact with the container 86, the liquid in the container 1 can be heated, and since the shut-off lid 86 is closed, the thermal efficiency at the time of heating is improved. , The liquid evaporated inside the lid 1a can be prevented from condensing and changing the composition of the liquid, and the effect of heat on the cold storage room 9 and the like can be reduced. Further, after the heater is stopped, if cooling air is blown from the air supply pipe 89 to the metal block 84, the metal block 84 can be cooled. Further, the container 1 is inserted into the container insertion hole 83, the air cylinder 85 is operated to close the shutoff lid 86, and the air cylinder 90 is closed.
To raise the closed container 87, and the upper surface 91 and the shutoff lid 86
Then, by operating the heater and sucking and exhausting the air inside the sealed container 87 through the vacuum pipe 88, the liquid in the container 1 can be quickly evaporated, so that the compact With such a configuration, the DNA sample can be easily dried.

As described above, since the liquid in the container 1 can be heated in a vacuum state, DNA drying by the ethanol precipitation operation can be performed in the incubator 10, and the sample preparation device can be downsized. In addition, since the air supply pipe 89 is provided, it is possible to smoothly perform the reaction from the primer annealing in which the reaction at the next different temperature level continues in a short time to the complementary strand synthesis reaction.

13 is a container reversing machine that fixes the container 1 with the lid 1a opened, inverts the container 1 in that state, and discards the liquid in the container 1.
82 is a holding table, 79 is a gear attached to the holding table 82, and the gear 79 is connected to a motor (not shown). Reference numeral 78 denotes a gear attached to the holding base 82, and the gear 78 meshes with the gear 79. Numeral 77 denotes a container fixing table fixed to the gear 78. The container fixing table 77 is a container that has a container 1 with a lid 1a opened and is mounted on the container 1 by applying a negative pressure to the inside of the container 1 by a negative pressure pipe (not shown). Fix 1 Reference numeral 80 denotes a reversing operation end point position specifying sensor provided on the holding table 82, and 81 denotes a reversing operation starting point position specifying sensor provided on the holding table 82, and includes gears 78 and 79, a container fixing table.
77 and the like constitute a container reversing machine 13.

In the reversing machine 13, the container 1 with the lid 1a opened is mounted on the container fixing table 77, and after the container 1 is fixed to the container fixing table 77, the motor is operated to rotate the container fixing table 77.
The liquid in the container 1 can be discarded.

Since the container reversing machine 13 can perform an operation of discarding a liquid of the order of several hundred μl in a short time, ethanol can be easily discarded in ethanol precipitation or the like. It is valid.

Reference numeral 16 denotes a container disposal hole for disposing of the used container 1.

14 is a container 1 for dispensing machine 7, centrifugal separator 11, mixer 12, insulated room 9, incubator 10, container reversing machine 13, container disposal hole
The transporter 14a for transporting between 16 is an X-axis drive mechanism, 93 is a motor of the X-axis drive mechanism 14a, 94 is a male screw, 95 is a coupling connecting the output shaft of the motor 93 and the male screw 94, and 14b is an X A Y-axis drive mechanism attached to the axis drive mechanism 14a, 14c is a vertical movement mechanism attached to the Y-axis drive mechanism 14b, 53 is an air cylinder of the vertical movement mechanism 14c, 53a is an air pipe of the air cylinder 53, and 14d is a vertical A gripping mechanism attached to the moving mechanism 14c, 54 is an air cylinder of the gripping mechanism 14d, 54a is an air pipe of the air cylinder 54, 55 is a handling chuck driven by the air cylinder 54, an X-axis driving mechanism 14a, a Y-axis driving The transfer device 14 is constituted by a mechanism 14b, a vertical movement mechanism 14c, and a gripping mechanism 14d.

In this transporter 14, if the X-axis drive mechanism 14a, the Y-axis drive mechanism 14b, and the vertical movement mechanism 14c are operated while the air cylinder 54 is operated and the container 1 is gripped by the handling chuck 55, the container 1 Dispenser 7, centrifuge 1
1. It can be transported between the mixer 12, the cool room 9, the incubator 10, the container reversing device 13, and the container disposal hole 16.

As described above, since the container 1 is grasped from the side by the handling chuck 55, the container 1 can be conveyed even when the lid 1a of the container 1 is open. Can be easily performed.

Further, a controller (not shown) for controlling the dispenser 7, the centrifuge 11, the mixer 12, the cool room 9, the incubator 10, the container reversing device 13, and the transfer device 14 is provided in the machine room 17. .

Furthermore, the distance between the position of the container mounting hole 20a at the transfer position of the container 1 of the transfer device 2 of the dispenser 7 and the transfer position of the container 1 of the mixer 12 and the transfer of the container 1 of the centrifuge 11 The distance between the entry position and the position where the container 1 is carried in / out of the mixer 12 is shorter than the distance between the position of the container mounting hole 20a and the position where the container 1 is carried in / out of the centrifuge 11.

In this way, the liquid dispensed by the dispenser 7 is
After mixing in 12, the time required for the operation of centrifuging in the centrifuge 11 can be shortened. Therefore, for example, when performing DNA purification operations such as phenol extraction and chloroform extraction in the process of preparing a DNA sample, first, phenol and chloroform are dispensed into the container 1 containing the DNA sample by the dispenser 7, and then the DNA After mixing the sample with the mixer 12, the sample is centrifuged by the centrifugal separator 11, and the aqueous layer containing DNA in the separated two layers is transferred to the transfer and dispenser 7.
The DNA purification operation can be performed in a short time.

Further, the distance between the position of the container mounting hole 20a and the position of loading / unloading of the container 1 in the cool room 9 is determined by the position of the container mounting hole 20a and the containers of the centrifuge 11, the mixer 12, the incubator 10, and the container reversing machine 13. 1 is shorter than the distance from the carry-in / out position.

In this way, the time required for transporting the container 1 stored in the cool room 9 to the dispenser 7 can be reduced. Therefore, for example, in the base sequence determination process, the operation of transporting the container 1 from the cold room 9 to the dispenser 7 and dispensing with the dispenser 7 is performed almost 100 times a day. The operation can be performed in a short time.

When preparing samples in a large number of containers 1, a large number of containers 1 are mounted on the centrifuge 11 after the number of containers 1 with the minimum number of centrifugations is set, and then the centrifuge 11 is operated. The time required for centrifuging the liquid in 1 can be reduced. For example, when performing the DNA purification operation as described above, the time required for one centrifugation is longer than the time required for dispensing, mixing, and transporting. If the centrifugal separator 11 is operated after being mounted on the separator 11, the time required for centrifuging the liquid in many containers 1 can be reduced.

Furthermore, when preparing the samples in many containers 1, the incubator 10 is operated after the number of containers 1 that minimizes the number of times of heating is mounted on the incubator 10, so that the liquid in many containers 1 can be prepared. Can be shortened in time required for heating. In the vacuum drying performed at the end of the ethanol precipitation, the lid 1a of the container 1 from which the DNA has been separated by the centrifugal separator 11 is opened by the lid opening mechanism 3, and the container 1 is inverted by the container inverting device 13, whereby the container 1 is inverted. After discarding the ethanol in 1, place the DNA in 1 in the incubator 10.
Is vacuum-dried to sufficiently remove ethanol, and then a buffer is poured into the container 1 by the dispenser 7 to dissolve the DNA, but the time required for vacuum drying in the incubator 10 is several minutes to several tens of minutes. The time required for opening the lid, reversing the container, and transporting the container is several seconds to several tens of seconds, while the number of containers required to minimize the number of times of vacuum drying when preparing the liquid in many containers 1 is minutes. If the incubator 10 is operated after the 1 is mounted on the incubator 10, the time required for vacuum-drying the DNA in many containers 1 can be reduced.

In the above embodiment, the cam mechanism 64 and the lever 6
6. Although the positioning mechanism is constituted by the rollers 65a to 65c, the pulse motor 67 and the like, the shaft 56 may be selectively connected to the positioning low-speed rotation motor or high-speed rotation motor using a clutch or the like. Further, in the above embodiment, the closed container 87
Is connected to the air supply pipe 89, the metal block 84 may be cooled by a Peltier element or a refrigeration cycle. Further, in the above-described embodiment, the vertical moving mechanism 14c having the air cylinder 53 is used, but a vertical moving mechanism having a motor may be used. In the above-described embodiment, the case where DNA purification operations such as phenol extraction and chloroform extraction are performed is described. However, ethanol precipitation and centrifugal acceleration for collecting the reaction solution at the bottom of the container 1 are also performed once. The time required for centrifugation is several minutes to several tens of minutes, while the time required for dispensing, mixing, and transporting is several seconds to several tens of seconds. If the centrifuge 11 is operated after being mounted on the centrifuge 11, the time required for centrifuging the liquid in many containers 1 can be reduced.

〔The invention's effect〕

In the nucleic acid sample preparation device according to the present invention, since the essential elements for the preparation of the nucleic acid sample are provided without excess and deficiency, the preparation of the nucleic acid sample can be easily performed.

 Thus, the effect of the present invention is remarkable.

[Brief description of the drawings]

FIG. 1 is a schematic perspective view showing a sample preparation device according to the present invention, FIG. 2 is a plan view showing a container transfer device around a dispenser,
FIG. 3 is a diagram showing a lid opening mechanism of the dispenser, FIG. 4 is a diagram showing a lid closing mechanism of the dispenser, FIG. 5 is a diagram showing a transfer dispenser of the dispenser, and FIG. Diagram showing the configuration of a pressure dispenser of the dispenser,
7 is a plan view showing a centrifuge, FIG. 8 is a sectional view showing a centrifuge bucket of the centrifuge, FIG. 9 is a view showing a mixer, FIG. 10 is a perspective view showing an incubator, FIG. FIG. 12 is a view showing a container reversing machine, FIG. 12 is a view showing a gripping mechanism of a transporter, and FIG. 13 is a graph showing a relationship between a centrifugal acceleration and a relative ratio of a DNA recovery amount in an ethanol precipitation operation. DESCRIPTION OF SYMBOLS 1 ... Container, 1a ... Lid 3 ... Lid opening mechanism, 4 ... Lid closing mechanism 5 ... Disposable tip, 7 ... Dispenser 8 ... Closed container, 9 ... Cold room 10 ... Incubator, 11 ... Centrifuge 12 ... Mixer , 13… Container reversing machine 14… Transporter 55… Handling chuck 62… Centrifugal bucket, 73… Container holding mechanism 76… Container direction correcting mechanism 86… Cut lid 88… Piping for vacuuming

Continuation of the front page (56) References JP-A-60-209177 (JP, A) JP-A 64-61667 (JP, A) JP-A 64-6759 (JP, A) JP-A 64-10178 (JP) JP-A-62-6171 (JP, A) JP-A-59-192963 (JP, A) JP-A-63-91565 (JP, A) JP-A-62-182665 (JP, A) 59-119267 (JP, A) JP-A-64-80865 (JP, A) JP-A-59-67285 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) G01N 35/00 -35/08

Claims (2)

(57) [Claims] 1. A container formed by integrally molding a fitable lid, a lid opening mechanism for opening the lid of the container, a lid closing mechanism for closing the lid of the container, and after opening the lid of the container. One or both of a dispenser for dispensing a liquid from the container to a separate container and a dispenser for dispensing a liquid sample from a predetermined sample bottle to the container, and the lid opening mechanism; Lid closing mechanism, a transport device that transports the container to the dispenser, a centrifuge that applies a centrifugal acceleration of 10,000 G or more to separate the liquid in the container, and a mixer that mixes the liquid in the container A cold storage chamber for storing the liquid in the container, an incubator for heating the liquid in the container, a container reversing device for discarding the liquid in the container, and a dryer for drying the liquid in the container, The above-mentioned container, the above-mentioned conveying device, the above-mentioned centrifugal separation Machine, the mixer, the cool room, the incubator, and a transporter for transporting between the dryers; the lid opening mechanism, the lid closing mechanism, the dispenser, the transporter, the centrifugal separator, and the mixing Machine, the cool room, the incubator, the container reversing machine, the drying machine, and a controller for controlling the transporter, the lid opening mechanism, the lid closing mechanism, the dispenser, the transporter, the transporter, The centrifugal separator, the mixer, the cold room, the incubator, the container reversing machine, the dryer, and the transporter are sequentially operated according to the process data stored in the controller, and the nucleic acid sample and the sample are operated. An apparatus for preparing a nucleic acid sample, wherein mixing, reaction, extraction and purification are performed automatically. 2. A container formed by integrally molding a fitable lid, a lid opening mechanism for opening the lid of the container, a lid closing mechanism for closing the lid of the container, and after opening the lid of the container. One or both of a dispenser for dispensing a liquid from the container to a separate container and a dispenser for dispensing a liquid sample from a predetermined sample bottle to the container, and the lid opening mechanism; Lid closing mechanism, a transport device that transports the container to the dispenser, a centrifuge that applies a centrifugal acceleration of 10,000 G or more to separate the liquid in the container, and a mixer that mixes the liquid in the container A cold storage chamber for storing the liquid in the container, an incubator for heating the liquid in the container, a container reversing device for discarding the liquid in the container, and a dryer for chaptering the liquid in the container. The container is transported by the transfer device, and the centrifuge is Machine, the mixer, the cool room, the incubator, and a transporter for transporting between the dryers; the lid opening mechanism, the lid closing mechanism, the dispenser, the transporter, the centrifugal separator, and the mixing Machine, the insulated room, the incubator, the container reversing machine, the dryer, a controller for controlling the transporter, a predetermined sample and a predetermined buffer solution held in a container in the insulated room, A plurality of reagents containing enzymes are transferred to the transfer device by the transfer device, the transfer device, the lid opening mechanism, the dispenser, the lid of the container is opened by a predetermined operation of a lid closing mechanism, The sample and the reagent are dispensed and mixed, the lid is closed, the container holding the dispensed mixed liquid is transferred to the mixer by the transfer device and mixed, and the container holding the mixed liquid is transferred to the transfer device. The first step of transporting and heating the incubator by heating, the sample liquid after the first step is transported to the transport device by the transport device, the transport device, the lid opening mechanism,
The container is opened by a predetermined operation of the dispenser and the lid closing mechanism, and the sample liquid and a predetermined salt, phenol and chloroform aqueous reagent are dispensed and mixed, and the lid is closed. The container holding the mixed and poured liquid is transported to the mixer by the transporter and mixed, and the container holding the mixed liquid is transported to the centrifuge by the transporter and centrifuged, The container holding the separated liquid is transferred to the transfer device by the transfer device, the lid is opened by the lid opening mechanism, the supernatant of the separated liquid is transferred to a new container by the dispenser, and A second process comprising closing the lid of the container holding the transferred liquid by the lid closing mechanism, and transferring the container holding the sample liquid after the first process or the second process to the transporter. The container is opened by a predetermined operation of the transfer device, the lid opening mechanism, the dispenser, and the lid closing mechanism, and the sample liquid, the predetermined salt, and the ethanol reagent are transferred to the container. Dispense and mix, close the lid, transport the container holding the dispensed and mixed liquid to the mixer by the transporter and mix, and transfer the container holding the mixed solution to the transporter And the container holding the separated liquid is transferred to the transfer device by the transfer device, and the lid is opened by the lid opening mechanism. Discarding the supernatant of the solution separated by the above, transporting the container holding the sample for which the supernatant has been discarded to the dryer by the transporter, drying the liquid remaining after the disposal, and drying the container. By the above conveyor After transferring the liquid to the transfer device and dispensing a predetermined liquid, the third step consisting of closing the lid by the lid closing mechanism, the preparation of a nucleic acid sample comprising one step or a combination of a plurality of steps is fully automated and performed. An apparatus for preparing a nucleic acid sample, which is performed sequentially.