JPH06327476A - Analyzer for gene - Google Patents

Abstract

(57) [Abstract] [Purpose] To provide an apparatus and a method for realizing highly reliable gene analysis by automatically and without interruption performing a gene analysis process that is extremely useful for gene diagnosis or medical treatment [Configuration] PCR A program equipped with a reaction tank and configured to search and determine the optimum reaction conditions enabling the determination of the DNA base sequence is executed, and PCR and the PCR before and after the PCR are performed using the optimum reaction conditions determined by the reaction condition search program. Carry out biochemical reactions. [Effect] Medically useful gene analysis processes such as DNA extraction from blood or other biological samples, purification, replication amplification of specific gene DNA, and enzymatic reaction treatment for detection are automatically executed without interruption, and good genes are always obtained. It becomes possible to prepare a pretreatment reaction product for analysis.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus and method for realizing gene analysis that is extremely useful for gene diagnosis or medical treatment.

[0002]

2. Description of the Related Art In recent years, the biological meanings of various genes have been clarified and their importance has been shown, and various genetic information analysis techniques for deoxyribonucleic acid (DNA), which carries genetic information, have been devised and improved. There is. Among them, the technique of DNA sequencing is a means for analyzing gene information in the most detailed manner, and high throughput is strongly desired. However, the reaction process involves cloning of a gene using an organism such as Escherichia coli, Due to the fact that DNA is extracted from the body, sequence reaction for sequencing, multi-steps, and organisms need to be handled, and the conditions of the entire reaction are very delicate, high throughput can be achieved by full automation. I didn't go.

However, Science (1985) vol. Three
7; p170 to p172. When PCR was invented by Saiki and the like, it became possible to replicate and amplify a target gene region without using organisms such as Escherichia coli. Proceeding National Academy of Sciences by G. Renren and Errich. S. A Vol. 85, 7652
-7656, 1988 (Ulf B. Gyllensten and He
nry A. Erlich Proceeding National Academy of Science
of USA Vol.85, p.7652-7656, 1988) and Vincent Murray's Nucleatic Acid Research, Vol. 17, pp. 8889 (Vincent Murray, Nuclea
PCR products directly as described in tic Acid Research).
A process was established in which all steps up to the sequence reaction, represented by the PCR direct sequence for A sequencing, were established and provided the basis for the full automation of DNA sequencing.

[0004]

However, in the PCR direct sequencing method, a side reaction product produced during PCR adversely affects the sequence reaction and obtains a DNA sequencing result with a good S / N ratio. There was a drawback that things were difficult.

In PCR, the annealing temperature, the denaturing temperature, the number of cycles, the amount of primer, etc. have a great influence on the production of main reaction products and side reaction products for the following reasons.

Since the amplification efficiency of the main reaction product decreases as the annealing temperature becomes higher than the optimum value, the required amount of reaction product is not produced under the annealing temperature condition that is too high. On the other hand, when the annealing temperature is lower than the optimum value, the amplification efficiency of the unnecessary side reaction product increases as the temperature lowers. Therefore, the generated side reaction product causes S of the sequencing result. / N ratio is deteriorated.

In PCR, double-stranded DNA is single-stranded DNA
Since the reaction starts from the point of dissociation (denature), the denatured temperature greatly affects the amplification efficiency of PCR. If this denaturation temperature does not reach the required temperature, the target main reaction product may not be obtained. On the other hand, however, if the denaturation temperature is excessively increased, the thermostable DNA polymerase, which is an essential enzyme for PCR, is inactivated by heat, and the amplification efficiency is also reduced.

The number of cycles is a parameter that determines the amplification factor of PCR. In PCR, the main reaction product is theoretically duplicated and amplified at an amplification factor of 2 times the cycle number, but as the amplification factor, that is, the number of cycles is increased,
Amplification of the main reaction product tends to saturate. This is a problem that cannot be avoided by PCR, because the generated main reaction product inhibits the subsequent main reaction.
On the other hand, the amount of side reaction products continues to increase at a constant rate, albeit slightly, so if the number of cycles is excessively large, the ratio of the amount of main reaction products / the amount of side reaction products decreases, and as a result, in the case of the direct sequencing method, DNA sequencing The resulting S / N ratio deteriorates.

The amount of primer affects the efficiency of the annealing reaction and the selectivity of the annealing reaction. When the amount of the primer is small, the amount of the main reaction product is small. On the other hand, when the amount of the primer is large, the selectivity during the annealing reaction is deteriorated, and as a result, the amount of the side reaction product is increased. That is, DN
The S / N ratio of the A sequencing result deteriorates. In particular, in the case of the direct sequencing method using asymmetric PCR, the amount ratio of primer amounts at the time of asymmetric PCR determines the amplification efficiency of the target single-stranded DNA, so an excessive amount of primer amount is required, This is also a delicate parameter, because if the temperature is exceeded, the production efficiency of the side reaction product also increases.

From the above reasons, it is understood that the optimum conditions for obtaining a good PCR product that can be subjected to direct sequencing exist in a very narrow range.

When carrying out the reaction manually by a skilled person,
A method in which the reaction products are separated and confirmed by gel electrophoresis in the middle of the reaction, the main reaction products and side reaction products are separated, and only the main reaction products are cut out from the gel and purified, if necessary. Has been solved by. However, in the case of an automatic device that continuously carries out the reaction by a certain process, it is difficult to perform such flexible operation, so it is necessary to search for optimum conditions.

Moreover, in the gene analysis, the reaction results such as the production efficiency of the main reaction product and the production efficiency of the side reaction product are different depending on the gene region of interest even if the processes are performed under the same reaction conditions. Since the optimum conditions differ depending on the region, it was very difficult to prepare a good sequence reaction product in a continuous process under constant conditions.

Therefore, medically useful gene analysis processes such as DNA extraction from blood or other biological samples, purification, replication amplification of specific gene DNA, and enzymatic reaction treatment for detection are automatically performed without interruption, and are always good. The present invention was carried out for the purpose of providing a gene analysis apparatus for preparing a reaction product for gene analysis.

In particular, to provide and carry out a genetic analysis process suitable for automation of preparing a good sequence reaction product in a continuous process.
It is also an object of the present invention to provide a fully automatic gene analysis apparatus for preparing a good sequence reaction product with good reproducibility.

[0015]

In order to achieve the above object, in the present invention, a reaction parameter of PCR is optimized according to a gene region of interest, and an apparatus for performing PCR using the identified optimum parameter is provided. It was constructed.

Specifically, a reagent dispensing mechanism, a centrifuge mechanism, a refrigerator and a freezer, a lid opening / closing mechanism of a plastic tube with a lid, a vortex mixer mechanism, and a decantation container inversion for extracting and purifying DNA from a biological sample. In addition to the mechanism and sample container transport mechanism, a temperature control block and temperature control mechanism that can control the reaction solution temperature for each sample, and a reagent injector that can set the reagent injection volume for each sample It was constructed so that the annealing temperature, the denaturation temperature, the number of cycles, and the amount of primer, which are the main parameters of PCR, can be changed for each sample.

Further, a simple gel electrophoresis apparatus for separating and detecting the PCR product, an ultraviolet transilluminator and a fluorescence detector were incorporated so that the amount of the reaction product in the PCR product could be detected if necessary.

Using these components, an optimum reaction condition is determined and stored, and an algorithm for performing gene analysis by an optimum process adopting the determined optimum condition is constructed and executed by the apparatus. did. That is, by calculating the main reaction product amount and the side reaction product amount from the pattern of the detected signal, by comparing and organizing the ratio of the main reaction product amount and the side reaction product amount for each reaction condition,
An apparatus was constructed to execute the program that determines the optimum reaction conditions. Furthermore, when a storage device that stores optimized parameters is incorporated and a gene region that has been optimized in the past is analyzed, it is possible to perform analysis using the stored parameters. I did.

[0019]

As in the present invention, the temperature of the reaction solution and the injection amount of the reagent can be set for each sample, so that the annealing temperature, the denaturation temperature, the number of cycles, and the amount of primer, which are important as parameters of PCR, At the same time, it will be possible to change it systematically and automatically. As a result, PC is indispensable for fully automated gene analysis.
It is possible to largely save the search for the R optimum condition.

Moreover, according to the present invention, by incorporating a simplified gel electrophoresis apparatus, a detection apparatus, and a reaction product amount analysis algorithm into an automatic apparatus, individual PCR products prepared by the optimum condition search program are separated by gel electrophoresis. Then, the amount of the main reaction product and the amount of the side reaction product are quantitatively analyzed by the analysis algorithm from the signal indicating the amount of the PCR product detected by the detection device, and the optimum condition can be automatically determined. That is, when a new gene region is analyzed, the optimal condition search program is executed, the reaction conditions determined to be optimal are then automatically adopted, and the sample preparation for the actual analysis is performed. Therefore, it becomes possible to prepare a sample for nucleotide sequence determination completely automatically.

The reaction conditions determined to be optimal are stored in a storage device, so that when analyzing a gene region that has been previously optimized, the optimum reaction conditions are stored in the storage device. After being read, it is possible to prepare a sample for nucleotide sequence determination according to the optimum reaction conditions.

The present invention realizes all pretreatment reactions starting from a biological sample such as blood or cells to a sequence reaction for nucleotide sequence determination. For example, the pretreatment process of medical gene analysis such as in the case of diagnosing an infectious disease by gene analysis is a fully automatic process such as extraction and purification of DNA from the above biological sample, selective amplification of a target gene, and enzymatic reaction treatment for detection. In most cases, it can be realized by the automatic device of the present invention.

In addition, since the present invention incorporates a simple gel electrophoresis apparatus and a detection apparatus, when the analysis is finished by separating and confirming the PCR product, for example, in the case of infectious disease diagnosis, It is possible to fully automate all the diagnostic processes from the pre-processing to the detection confirmation in one housing.

[0024]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a block diagram showing the device configuration of the present invention.
2 is a schematic perspective view showing the internal structure of an embodiment of the gene analysis device according to the present invention, FIG. 3 is a plan view showing a delivery device for a dispensing element, and FIG. 4 is a diagram showing a lid opening mechanism for the dispensing element. , Fig. 5
6 is a view showing a lid closing mechanism of a dispensing element, FIG. 6 is a view showing a transfer dispensing machine and its peripheral mechanism, FIG. 7 is a view showing a configuration of a pressure feeding dispenser, and FIG. 8 is a plan view showing a centrifugal separation element. FIG. 9, FIG. 9 is a cross-sectional view showing a centrifuge bucket of a centrifuge of centrifugal separation elements, FIG. 10 is a view showing mixing elements, FIG. 11 is a perspective view showing heating and vacuum drying elements, and FIG. 12 is a container inversion element. FIG. 13 is a view showing a gripping mechanism portion of the carrying element, FIG.
FIG. 15 is a diagram showing the treatment of waste, FIG. 15 is a simplified gel electrophoresis element for confirming separation and quantification of PCR products, and FIG. 16 is detected when the PCR products are electrophoresed by the gel electrophoresis tank of the present invention. FIG. 17 is a diagram showing a signal pattern, FIG. 17 is a diagram showing a gene pretreatment process adopted in this example, and FIG. 18 is a PCR based on various conditions for optimization condition search, and a simple electrophoresis apparatus is used. It is a figure showing an example of a result of having analyzed by gel electrophoresis.

First, the overall device configuration of an embodiment of the present invention will be described with reference to a block diagram (FIG. 1). In the device body,
Dispensing element 154, container conveying element 14, centrifuge element 1
1, a mixing element 12, a container reversing element 13, a heating, a vacuum drying element 10, and a gel electrophoresis element 131, each of which is controlled by a computer 149 of the apparatus body control unit. The dispensing element 154 includes a transport mechanism 2, a lid opening / closing mechanism 3, 4, a transfer dispensing mechanism 7, and a pressure-feeding dispensing mechanism 21,
The heating and vacuum drying element 10 has a temperature control block 84,
A vacuum chamber opening / closing mechanism 87 is included. Gel electrophoresis element 1
The detection result of 31 is sent to the electrophoresis result analysis device 151, analyzed by an algorithm to determine the optimum condition, and stored in the storage device 152. The stored optimum conditions are retrieved as needed by the operator,
It is sent to the device body control unit 149 and adopted as the reaction condition of the optimization process. The operator terminal 150 is
It is in communication with the apparatus main body control unit 149 and the storage device 152, and is configured to check the execution status of the process, interrupt the process, and retrieve parameters from the storage device according to the operator's judgment.

A liquid dispensing operation into the container 1 according to the present invention will be described with reference to FIGS. 2, 3, 4, 5, 6 and 7.

The container 1 used in the present invention is provided with a lid 1a which can be fitted as shown in FIG. 4, and includes the container 1 and the lid 1a.
Are integrally molded, and the container 1 is made of a lightweight and strong plastic such as polypropylene, and the container 1 can withstand a centrifugal acceleration of 10,000 G or more.

In FIG. 2, reference numeral 7 denotes a transfer / dispensing mechanism, which dispenses a liquid such as a reagent into the container 1. A transport mechanism 2 transports the container 1 on a plane. A lid opening mechanism 3 opens the lid 1a of the container 1 transported by the transport mechanism 2. A lid closing mechanism 4 closes the lid 1a of the container 1 transported by the transport mechanism 2. 21a is a pressure-dispensing dispenser 2
1 is a liquid pipe (see FIG. 7), 8 is a closed container, and the liquid pipe 21 a and the closed container 8 are connected through the inside of the machine room 17.

The detailed structure of the transport mechanism 2 is shown in FIGS. 3, 2a and 2b are turntables of the transport mechanism 2, and the pulse motors 18a and 18 in FIG.
The turntables 2a and 2b are driven by b. 20
Is a container mounting hole provided in the turntables 2a and 2b. The arrows indicate the rotation directions of the turntables 2a and 2b.

The detailed structure of the lid opening mechanism 3 is shown in FIG.
The container mounting hole 20 has a lever 24 and an elastic member 2 for pushing the lever 24.
3 is provided to fix the container 1 to the container mounting hole 20. The lid opening mechanism 3 is configured on a holding table 29 fixed to a base 30. The gear 28a is driven by the motor 120,
The gear 28a and the gear 28b are connected via a belt 28c. Further, the gears 27a and 27b mesh with the gear 28b, and the lid hooking member 25 is rotatably attached to the gears 27a and 27b by the pins 26a and 26b. 25a is the tip of the hooking member 25.
The tip portion 25a has a shape for hooking the lid 1a.

The detailed construction of the lid closing mechanism 4 is shown in FIG.
The lid closing mechanism 4 is configured on a holding table 35 fixed to the base 30. The gear 34a is driven by the motor 121, and the gear 34a and the gear 34b are connected via the belt 34c.
Are connected. Further, the gears 33a and 33b are the gears 34
The lid pushing down member 31 is rotatably attached to the gears 33a and 33b by the pins 32a and 32b, and the lid pushing down member 31 is provided with a roller 31a. The roller 31a pushes the lid 1a. Has become.

The detailed structure of the transfer and dispensing mechanism 7 is shown in FIG. Reference numeral 5 is a dispensing tip with a filter (hereinafter, abbreviated as a dispensing tip), which is attached to the tip mounting portion 38b. Although illustration is omitted, it is composed of a chip body portion and a filter portion. Reference numeral 52 denotes a holding table, which is fixed to the base 30, and the transfer / dispensing mechanism 7 is constructed based on the holding plate 50 so as to be movable on the holding table 52. The holding plate 50 is moved on the holding table 52 in the left-right direction on the paper surface by a driving device including a motor 51a and a belt 51b. Reference numeral 49 is a pulse motor attached to the holding plate 50, 47b is a male screw connected to the pulse motor 49, and a female screw 47a screwed to this is fixed to the holding plate 46, and the holding plate 46 is attached to the holding plate 50. It is attached so that it can move up and down. The holding plate 50 is provided with a position sensor (not shown) for designating the origin and the vertical limit of the vertical movement of the holding plate 46. Reference numerals 37a and 37b denote body portions attached to the holding plate 46. The body portions 37a and 37b and the tip attachment portions 38a and 38b include plungers 36a and 36b (not shown), and the plunger portion 36a included in the body portion 37a. The diameter of the plunger 3 is included in the body portion 37b.
It is smaller than the diameter of 6b. 44 is a pulse motor attached to the holding plate 46, and 42b is a male screw connected to the pulse motor 44.
a is screwed, and covers 39a, 39b and plungers 36a, 36b are attached to the female screw 42a, and these are vertically movable with respect to the holding plate 46. 40
Reference characters a and 40b are tip removing members for removing the dispensing tips 5 attached to the tip attaching portions 38a and 38b by being pushed by the covers 39a and 39b. The positions of the covers 39a, 39b and the plungers 36a, 36b are designated by the position sensor (not shown) provided on the holding plate 46 as the origin / upper limit. Reference numeral 6 is a tip supply mechanism for supplying the dispensing tip 5, 6a is a storage portion for storing the dispensing tip 5, 6b is a transport mechanism for transporting the storage portion 6a, 6c is a motor, and the storage portion 6a, the transport mechanism 6b, and the motor 6c constitute the chip supply device 6. Reference numeral 15 denotes a chip disposal hole provided between the transport mechanism 2 and the chip supply device 6.

The detailed structure of the pressure-dispensing dispenser 21 is shown in FIG. Reference numeral 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, and 21f is an air tank 21e. Connected pressure regulating valve, 21i is a high pressure air source connected to pressure regulating valve 21f, 21d is a solenoid valve connected to solenoid valve 21c, 21g
Is an atmospheric pressure source connected to the solenoid valve 21d, 21h is a vacuum pump connected to the solenoid valve 21d, and 21a has a liquid 8 at one end.
a liquid pipe that is immersed in a and has the other end located above the turntable 2b as shown in FIGS. 3 and 6,
A pressure-dispensing dispenser 21 is configured by the closed container 8a, the solenoid valve 21c, the vacuum pump 21h, and the like. In this device, five sets of the pressure-dispensing dispenser 21 are installed, and different liquids are placed in each closed container 8. I am putting it in.

The dispensing operation is carried out by the operations of the transfer dispensing mechanism 7, the transport mechanism 2, the lid opening mechanism 3, the lid closing mechanism 4, and the pressure feeding mechanism 21. The operation of each mechanism will be described below.

In FIG. 6, pulse motors 18a and 18b are provided.
3 is turned, the turntables 2a and 2b are rotated in the direction of the arrow in the drawing of FIG. 3, so that the container 1 placed in the container placement hole 20 can be conveyed on a flat surface. Then container 1
Is positioned on the lid opening mechanism 3, and the container 1 is fixed to the container mounting hole 20 by the lever 24 and the elastic member 23 that pushes the lever 24, and then the gear 28 is rotated clockwise by the motor 120 as shown in FIG. When driven to, the gears 27a and 27b rotate counterclockwise in FIG. 4 and the tip portion 25a moves upward, so that the lid 1a of the container 1 can be opened.

Then, when dispensing the liquid, use this lid 1
20a, 20c shown in FIG. 3 with the container a open.
Located in. Then, in FIG. 6, by driving the motor 51a and moving the holding plate 50, the chip mounting portion 38
The positions of a and 38b in the horizontal direction of the paper surface of FIG. 6 can be determined, and by operating the pulse motor 49, the positions of the chip mounting portions 38a and 38b in the vertical direction can be determined. If the pulse motor 44 is activated, the plunger 3
The vertical positions of 6a, 36b and covers 39a, 39b can be determined.

Therefore, the tip feeding mechanism 6 is driven to move the dispensing tip 5 to the feeding position, and the tip mounting portion 38a,
If the tip mounting portions 38a and 38b are lowered after the 38b is positioned above the storage portion 6a, the tip mounting portion 38a
The dispensing tip 5 can be attached to either one of a and 38b, and then the tip attaching portions 38a and 38b are moved up and moved horizontally to move the tip attaching portions 38a and 38b.
Position above the container mounting hole 20b at the dispensing position,
Dispense tip 5 by lowering tip attachment parts 38a, 38b
Is immersed in the liquid in the container 1 placed in the container placement hole 20b, the plungers 36a, 36b are raised to suck the liquid into the dispensing tip 5, and the tip mounting portions 38a, 38
b is moved up and moved horizontally to move the chip mounting portion 38a,
38b is positioned above the container mounting hole 20c at the dispensing position of the turntable 2b, and the chip mounting portions 38a,
8b to lower the tip of the dispensing tip 5 into the container mounting hole 20c.
Inserted into the container 1 placed on the
When the liquid in the dispensing tip 5 is discharged by moving down the container 6b, the container 1 placed in the container mounting hole 20b is moved to the container mounting hole 2
The liquid can be dispensed into the container 1 placed at 0c.

Next, the chip mounting portions 38a and 38b are lifted and moved horizontally to position the chip mounting portions 38a and 38b above the chip disposal hole 15, and the covers 39a and 3b.
When 9b is lowered, the tip removing members 40a and 40b are pushed by the covers 39a and 39b, the dispensing tip 5 is removed from the tip mounting portions 38a and 38b, and the dispensing tip 5 is discarded in the tip discarding hole 15. .

In this case, when the target dispensing amount is small,
The dispensing tip 5 is attached to the tip attaching portion 38a, and when the target dispensing amount is large, the dispensing tip 5 is attached to the tip attaching portion 38b to perform suction and discharge operations of the liquid. Here, if the dispensing operation is performed without the filter of the dispensing tip 5, the sample becomes an aerosol and the tip mounting portion 38
Even if the dispensing tip 5 is attached to the inside of a and b, and then the dispensing tip 5 is replaced with a new one, when dispensing to another sample, the tip mounting portion 3
8. The aerosol attached inside adheres to the sample. In the present invention, since the filter is provided, the aerosol containing the sample is collected by the filter and does not enter the tip mounting portion 38. Therefore, it is possible to prevent the sample from mixing with other samples by exchanging the dispensing tip 5 for each dispensing operation.

Next, the operation of the pressure-feeding and dispensing mechanism 21 will be described. In FIG. 3, the container 1 is conveyed to a position below the liquid pipe 21a on the turntable 2b with the lid 1a opened, and the closed container 8 and the atmospheric pressure source 21g are connected by the solenoid valves 21c and 21d in FIG. Solenoid valve 21
If the closed container 8 and the high-pressure air source 21i are connected by switching c, the liquid 8a from the closed container 8 through the liquid pipe 21c.
Is supplied into the container 1. From this state, the solenoid valve 21
Switching between c and 21d, the closed container 8 and the vacuum pump 21h
After connecting and, the solenoid valve 21d is switched to close the sealed container 8
By connecting this to the atmospheric pressure source 21g, the liquid 8a in the liquid pipe 21a can be completely drawn back into the closed container 8.

As described above, the transfer / dispensing mechanism 7 accurately dispenses a small amount of liquid of the order of several μl, and the pressure-dispensing / dispensing mechanism 21 can inject a large amount of liquid in a short time.

Next, the operation of the lid closing mechanism 4 will be described. When the gear 34 is driven clockwise in FIG. 5 by the motor in FIG. 5, the gears 33a and 33b rotate counterclockwise in FIG. 5, and the roller 31 moves in an arc, so that the container 1
The lid 1a can be closed.

The centrifugation operation of the reagent in the present invention will be described. Reference numeral 11 in FIG. 2 denotes a centrifugal separation element, and FIG.
FIG. 9 shows its detailed configuration. The centrifuge element 11 is a centrifuge that separates the liquid in the container 1 by applying a centrifugal acceleration, 68 is a main body of the centrifuge 11, 56 is a shaft directly connected to a high-speed rotation motor (not shown), and 57 is a shaft 56. Mounted on the rotor, 58 is the outer wall of the rotor 57, FIG.
69 is a steel wire attached to the rotor 57, 62 is a swing rotor type centrifugal bucket supported by the steel wire 69, and 59 is a container insertion hole provided in the centrifugal bucket 62. As shown in FIG. Is inserted. Reference numeral 60 denotes a groove provided in the centrifugal bucket 62, and the groove 60 is engaged with the upper portion of the container 1. 61 is a rotor 57 and a centrifuge bucket 62
A gap adjusting plate provided between the main body 68 and the gap adjusting plate 64, and a cam mechanism 64 a rotatably attached to the main body 68.
, 63 is an air cylinder for rotating the cam mechanism 64, 66 is a lever rotatably provided on the main body 68, and 65a to 65c are rollers attached to the lever 66. .About.65c are pressed against the cam surface 64a by an elastic member (not shown). A pulse motor 67 rotates the roller 65a.
7 is provided with a photo sensor (not shown) for detecting the position of the cam mechanism 64, the lever 66, the roller 65a.
The positioning mechanism is configured by 65c, the pulse motor 67, and the like.

In this centrifuge 11, if the container 1 is inserted into the container insertion hole 59 as shown in FIG. 9, the upper projection of the container 1 is engaged with the groove 60, and the high speed rotation motor is operated. The rotor 57 is rotated at a high speed such that the centrifugal acceleration applied to the container 1 is 10,000 G or more, the centrifugal bucket 62 is rotated counterclockwise around the steel wire 69 in FIG. The liquid in 1 can be separated.

After stopping the high speed rotation motor,
If the air cylinder 63 is reduced, the cam mechanism 64 rotates, the rollers 65a to 65c are pushed by the cam surface 64a,
The rollers 65a to 65c contact the outer wall 58. In this state, if the pulse motor 67 is operated according to the output of the photo sensor, the centrifugal bucket 62 can be brought into and out of the container 1. After this, the air cylinder 63
The cam mechanism 64 rotates, the rollers 65a to 65c are pressed against the cam surface 64a by the elastic member,
The state shown in FIG. 8 is obtained.

Thus, the centrifugal acceleration applied to the container 1 is 1
Since the rotor 57 rotates at a high speed of 0000 G or more, it is possible to increase the DNA recovery rate in the ethanol precipitation operation of dispensing ammonium acetate and ethanol or sodium acetate and ethanol into the DNA solution, mixing and centrifuging. .

The operation of mixing the liquid in the container 1 according to the present invention will be described. Reference numeral 12 in FIG. 2 denotes a mixing element,
FIG. 10 shows the detailed configuration. 70 is a mixing element body, 74 is a vortex mixer, and 72a to 72c are gears attached to the mixing element body 70.
2c is engaged. 72d is a lever fixed to the gear 72a, 71 is an air cylinder having one end attached to the mixing element body 70, and the other end of the air cylinder 71 is pin-connected to the lever 72d. 73 is a container pressing mechanism fixed to the gear 72c, 76 is a container direction correcting mechanism for correcting the direction of the container 1 by pushing the protruding portion of the container 1,
Reference numeral 75 is an air cylinder 75 for driving the container direction correcting mechanism 76. The air mixing cylinder 75 includes a vortex mixer 74, a container pressing mechanism 73, a container direction correcting mechanism 76, and the like.
Is configured.

In the mixing element 12, after the container 1 is attached, the air cylinder 71 is operated, the container pressing mechanism 73 is pressed to hold the container 1, and then the vortex mixer 74 is operated. 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.

A method of keeping liquid cold in the present invention will be described. In FIG. 2, 9 is a cold storage room for storing the liquid in the container 1, 9a is a cold storage room, and 9b is a freezing storage room. The upper parts of the cold storage room 9a and the cold storage room 9b are engaged with the upper part of the container 1. A groove is provided, and a cold storage room 9a and a freezing storage room 9b constitute a cold storage room.

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

The method for heating the liquid in the present invention will be described. Reference numeral 10 in FIG. 2 denotes a heating element for heating the liquid in the container 1, and FIG. 11 shows its detailed configuration. Figure 1
In 1, the numeral 87 is a closed container, 91 is the upper surface of the closed container 87, and 88 is connected to the closed container 87 by a vacuum pipe.
The other end is connected to a vacuum pump (not shown), and an air suction / exhaust unit for suctioning / exhausting the air inside the closed container 87 is configured by a vacuum sweeping pipe 88 or the like. A refrigerant supply pipe 89 is connected to the closed container 87. Reference numeral 84 denotes a metal block, which is provided in the closed container 87 and has 16 container insertion holes 83.

The metal block 84 is heated by a heater (not shown), but a total of four heaters having different container insertion holes 83 and four different refrigerant supply conduits 89 are provided. Platinum resistance wire temperature sensor 1
32 is provided, and it is possible to control the sample liquid temperature to a different temperature for every 4 samples.

The upper surface of the metal block 84 is provided with a groove with which the upper portion of the container 1 is engaged, so that the container 1 can be positioned. The shape of the container insertion hole is the container 1
It is cut according to the shape of, and the surface of the container is shaped with a high thermal conductive adhesive so as to make surface contact with the container 1. Because of this, the metal block and container 1 and container 1
Sufficient thermal contact is maintained with the reaction solution via the. 8
6 is a blocking lid, which is rotatably attached to the closed container 87. An air cylinder 85 opens and closes the blocking lid 86. 90 is an air cylinder for moving the closed container 87 up and down.

In the heating element 10, the container 1 is inserted into the container insertion hole 83, the air cylinder 85 is operated to close the shutoff lid 86, the air cylinder 90 is operated to raise the closed container 87, The liquid in the container 1 can be heated for an appropriate time by operating the heater after the upper surface 91 and the blocking lid 86 are brought into contact with each other, and since the blocking lid 86 is closed, The thermal efficiency is improved, the temperature difference between the inside of the container 1 and the wall surface of the container is reduced, and it is possible to prevent the evaporated liquid from aggregating inside the lid 1a and changing the density of the liquid. The influence of heat given can be reduced.

After stopping the heater, the refrigerant supply pipe 8
By circulating the coolant from the inside of the metal block 84, the metal block 84 can be cooled. Further, after the cooling reaches a predetermined temperature, if the circulation of the refrigerant and the heater heating are operated in competition with each other, the liquid temperature can be reached to the predetermined temperature in an appropriate time.

In this way, since the temperature of the liquid in the container 1 can be continuously operated to warm, cool and reheat,
It is possible to perform PCR in which a gene is amplified by continuously performing an enzymatic reaction in a nucleic acid sample. Moreover, by controlling different temperatures for every 4 samples, PCR of 4 conditions can be performed simultaneously.
Can be done. When the PCR cycle number condition is changed, the sample after a predetermined cycle number is moved from the PCR reaction tank 10 to the refrigerator by the container transport mechanism to carry out the reaction at different cycle numbers.
CR products can be made.

Further, the container 1 is inserted into the container insertion hole 83, the air cylinder 85 is operated to raise the closed container 87, the upper surface 91 and the blocking lid 86 are brought into contact with each other, and then the heater is operated and If the air inside the closed container 87 is sucked and exhausted through the vacuum tubing 88, the liquid in the container 1 can be evaporated quickly, so that the DNA sample can be easily dried with a compact structure. it can.

The liquid removing method according to the present invention will be described. Reference numeral 13 in FIG. 2 denotes a container reversing machine, which is a lid 1
The container 1 in which a is opened is fixed, and the container 1 is inverted in that state to remove the liquid in the container 1. FIG. 12 shows its detailed configuration. In FIG. 12, reference numeral 82 denotes a holder, 79 denotes a gear, which is attached to the holder 82 and is connected to a motor (not shown). Reference numeral 78 denotes a gear, which is attached to the holding base 82 and meshes with the gear 79. Reference numeral 77 denotes a container fixing base, which is fixed to the shaft of the gear 78, and after the container 1 having the lid 1a opened is mounted, the container 1 is fixed by making the inside a negative pressure by a negative pressure pipe (not shown). . A reversing operation end point position designation sensor 80 is provided on the holding table 82.
Reference numeral 81 denotes a reversing operation start point position specifying sensor, which is provided on the holding table 82.

In the reversing element 13, the container 1 having the lid 1a opened is mounted on the container fixing base 77, the container 1 is fixed to the container fixing base 77, and a motor (not shown) is operated to operate the container. The liquid in the container 1 can be removed by rotating the fixing table 77.

In FIG. 2, reference numeral 16 denotes a container discarding hole, in which the used container 1 is discarded.

A method of transporting the container 1 according to the present invention will be described. In FIG. 2, reference numeral 14 denotes a conveying element, and the container 1
The transfer device 2, the centrifugal separation element 11, the mixing element 12, the cold storage chamber 9, the heating element 10, the container inverting element 13, and the container disposal hole 1.
Transport between 6 14 a is an X-axis drive mechanism, 93 is a motor of the X-axis drive mechanism 14 a, 94 is a male screw, and 95 is a coupling, which connects the output shaft of the motor 93 and the male screw 94. 14b is a Y-axis drive mechanism, and is attached to the X-axis drive mechanism 14a. A vertical movement mechanism 14c is attached to the Y-axis drive mechanism 14b. FIG. 13 shows the detailed structure of the gripping mechanism for gripping the container 1 of the transport element 14. 14
A gripping mechanism d is attached to the vertical movement mechanism 14c. An air cylinder 53 moves the gripping mechanism 14d up and down by the air from the air pipe 53a. An air cylinder 54 drives the handling chuck 55 by the air from the air pipe 54a.

In this carrying element 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 is conveyed device 2, centrifuge element 11, mixing element 12, cold storage chamber 9, heating element 10, container inverting element 13, container waste hole 16
The container 1 can be conveyed in between.

Processing of waste liquid and waste in the present invention will be described. In FIG. 14, 104a is a waste liquid, which is discharged by the operation of the container reversing machine 13. 104b
Is a waste liquid receiver, 104c is a waste liquid bottle, and temporarily stores the waste liquid 104a. A waste liquid pipe 104d connects the waste liquid receiver 104b and the waste liquid bottle 104c. 16a is a container waste pipe,
It is connected to the container disposal hole 16. A sterilizer 105 is connected to the container waste pipe 16a. 15a is a chip disposal pipe,
One is connected to the tip disposal hole 15 and the other is connected to the sterilizer 10.
Connected to 5.

A simple gel electrophoresis device and a fluorescence detection device incorporated in the automatic device of the present invention will be described.
In FIG. 15, 132 is an electrophoresis tank, 134 and 13
5 is an anode and cathode electrode, 136 is a gel holder, 137
Is an agarose gel for electrophoresis, 140 is a sample introducing groove (well) opened on the gel, and 138 is a UV transilluminator to irradiate the gel with ultraviolet rays of a short wavelength. 139 is a fluorescence detector with a filter, in which ethidium bromide is intercalated by a photodiode.
It is fluorescence when excited by short-wavelength ultraviolet light with respect to A. 5
Fluorescence around 90 nm is detected. 143 and 144 are quartz glass plates, which are the side of the gel on which the detector is arranged and the UV.
It is provided on the bottom surface of the gel where the transilluminator is located. As shown in FIG. 2, in an actual electrophoresis and detection state, the upper part is covered with a lid 142 having a sample supply hole 141 at a position corresponding to the well 140 opened on the gel for light shielding. However, it is shown here with the lid removed.

Ethidium bromide was contained in the gel and the buffer solution for electrophoresis at about 5 μg / ml and 0.5 μg / ml, respectively.
It has been added to the concentration of. When the PCR product is used for detection, the transfer and dispensing mechanism 7 shown in FIG. 6 is used.
Then, the PCR product solution is introduced into the well 140. That is, the tip supply mechanism 6 is driven to move the dispensing tip 5 to the supply position, and the tip attachment portions 38a and 38b are moved to the storage portion 6a.
Chip mounting portions 38a, 38b after being positioned above
And the dispensing tip 5 is attached to the tip attaching portion 38a. Thereafter, the chip mounting portions 38a, 38b are raised and moved horizontally to position the chip mounting portion 38a above the container mounting hole 20b at the dispensing position on the turntable, and the chip mounting portions 38a, 38b. Container 1 in which the tip of the dispensing tip 5 is placed in the container placement hole 20b
It is immersed in the liquid inside, and the plungers 36a and 36b are raised to suck the liquid into the dispensing tip 5. After that, the chip mounting portions 38a, 38b are raised and moved horizontally to position the chip mounting portions 38a, 38b above the sample supply hole 141 at a predetermined position of the simplified gel electrophoresis tank. 38a and 38b are lowered to insert the tip of the dispensing tip 5 into the well 140 at a predetermined height, and the plungers 36a and 36b are lowered to discharge the liquid in the dispensing tip 5. In this way, the PCR product solution can be dispensed from the container 1 placed in the container placing hole 20b to a predetermined well in the gel for electrophoresis, and then a voltage of 100 V to 200 V is applied between the electrodes 134 and 135. PC by adding
The R products can be separated by electrophoresis in an agarose gel.

Since the major PCR products belong to a single band or two bands in the case of asymmetric PCR by gel electrophoresis, even if the influence of side reaction products is taken into consideration, an interval of about 10 minutes or more is obtained. If the PCR product is opened by introducing the PCR product into the gel, the bands derived from different samples will not be crossed and thus will not be indistinguishable. That is, there is no problem even if all gel electrophoresis for optimization is continuously performed with one gel.

However, gel electrophoresis and detection for determining the optimum parameters do not necessarily have to be performed in the apparatus, and the products prepared by the apparatus of the present invention under various conditions can be manually operated at once. It is also possible to perform electrophoresis and input the optimum parameter values into the storage device 152 at the operator's discretion.

The amount of PCR products separated by electrophoresis is quantitatively detected as fluorescence intensity by ethidium bromide by a fluorescence detector with filter 139 provided at a predetermined position. A photodiode is used as a detector, and the entire gel is excited from below by a UV transilluminator 138 as shown in FIG. 15, and the fluorescence in the horizontal direction, which is the vertical direction, is detected by the photodiode. The position of the photodiode in the migration direction is configured so that it can be changed as necessary (not shown).

The method for analyzing the amount of PCR product detected in the present invention will be described. As described above, when the PCR product is electrophoretically separated by a simplified gel electrophoresis bath, a peak pattern as shown in FIG. 16 is obtained. This peak pattern is analyzed by the following algorithm to calculate the amount of main reaction product and the amount of side reaction product in the peak.

First, the signal intensity before a peak is detected for a predetermined time from the start of electrophoresis is stored as a background signal (background noise), and the average value of the background noise is set to a reference value (0 point). ). PCR to be detected after processing this background noise
The output signal of the detector 139 is sampled at a predetermined period and stored in the storage device 152 for a predetermined time required for the product to pass by the electrophoresis in front of the detector 139. The stored signal data is processed as a significant signal from the time when a signal having a predetermined amplitude or more with respect to the amplitude of background noise is detected. This treatment is carried out for each reaction product sample introduced into the electrophoresis gel 137 one after another,
It is cut out for each electrophoretic sample. FIG. 16 shows an example of the peak waveform of the cut out electrophoretic sample. As shown by the solid line in the example of the figure, the resulting signal is
First, a peak due to the primer appears, and then a peak corresponding to the amount of the main reaction product appears, and with this decay, the signal due to the side reaction product generally appears in the added form.

The signal peak waveform to be processed is first subjected to smoothing processing to remove abnormal values due to noise.
Then, the time (average value tm) that gives the maximum value of the highest peak corresponding to the amount of the main reaction product and the standard deviation σ of the signal to be processed (corresponding to the time when the inflection point of the highest peak is obtained) are calculated. Originally, it is approximated by a Gaussian error function indicated by a thick broken line. And tm of this approximated error function
The integrated value of ± 2σ is the main reaction product amount. Next, a value obtained by subtracting 1/2 of the corresponding main reaction product amount from the integrated value after the time (tm) giving the average value of the approximated error function in the entire cut out waveform is stored as the side reaction product amount. . The reason why the integration range is after tm is that the signals before tm-2σ are mainly derived from the primer and the signals of the side reaction products are mainly detected after tm.

The main reaction product amount and the side reaction product amount analyzed as described above are arranged for each parameter, and the ratio (S / N ratio) between the main reaction product amount and the side reaction product amount is maximized. The optimum condition is determined by the algorithm of the present invention for determining the optimum condition.

The parameters determined as the optimum conditions are the storage device 152 together with the gene region name, starting material, starting material amount, date and time, history of the optimum value search program, and the like.
When the same gene region is stored in (FIG. 1) and is analyzed, the optimum parameter corresponding to the gene region is read from the storage device as needed, and the analysis reaction is advanced by the parameter. There is.

The embodiments shown above are merely examples of the present invention, and the above embodiments do not limit the present invention. For example, in the present Example, a submarine-type agarose electrophoresis apparatus is used for the electrophoretic separation of PCR products. However, this may be performed by another electrophoretic separation such as a method of performing electrophoretic separation by a gel packed in a capillary tube. I don't mind.

One example of the present invention will be described below by exemplifying a case where polymorphism analysis of the human leukocyte antigen (HLA) gene DQα region is carried out using the whole blood as a starting material by applying the apparatus and process of the present invention.

FIG. 17 shows the gene pretreatment process adopted in this example.

A process for extracting and purifying DNA from a biological sample will be described. Starting material is 50 μl of whole blood in container 1
(1.5 ml) 1 ml of distilled water
Is added by the pressure-feeding dispenser 21 and shaken by the mixing element 12 to hemolyze. After centrifuging this solution at 10,000 × g for 1 minute, the supernatant is removed (decantation) by the inverting element 13 to obtain a precipitate. Distilled water was added to this again, and the same hemolysis, centrifugation, and decantation were performed again to obtain a precipitate obtained by performing hemolysis twice. TNE in this precipitate
Buffer (10 mM Tris-HCl (pH 8.
0), 1 mM EDTA (pH 8.0), 100 mM N
aCl) 90 μl, 10% SDS 10 μl, 10 m
5 g / ml of Protenase K (MERCK)
Add μl and incubate at 50 ° C. for 10 minutes to decompose the protein.

Next, a phenol / chloroform solution (water-saturated phenol: water-saturated chloroform (1
/ 24 addition of isoamyl alcohol) = 1: 1) is 100
μl was added and mixed and stirred by the mixing element 12, and then the supernatant (water layer portion) of the product centrifuged at 10000 × g for 1 minute was sucked by the dispensing element 154 and transferred to another container 1 (phenol / chloroform extraction). ). After performing this phenol-chloroform extraction twice, 30% of the amount of water layer
(Volume ratio) amount of 3M NaCl aqueous solution and aqueous layer amount (NaC
1) containing 2.5 times the amount of 99.5% ethanol,
Centrifuge at 10,000 × g for 20 minutes (ethanol precipitation).

After centrifugation, the supernatant portion was decanted by the container inverting element 13, and 200 μl of 80% ethanol aqueous solution was gently added to the pellet-like material (DNA) precipitated on the bottom wall of the container at 10,000 × g. After centrifugation for 3 minutes, the supernatant portion is decanted by the container inverting element 13 and removed (ethanol rinse). This ethanol rinsing operation was repeated twice, and the container 1 containing the pellet-shaped precipitate was heated with the lid open.
0 into the container insertion hole 83 and the vacuum drying elements 86 and 87 evaporate the remaining ethanol aqueous solution.
Obtain NA pellets.

After drying, the DNA pellet was mixed with 100 μl of TE buffer solution (10 mM Tris-HCl (pH
7.5), dissolved in 1 mM EDTA) (end of DNA extraction and purification steps).

The PCR amplification step of the target DNA portion will be described. 50 mM KCl as PCR buffer,
10 mM Tris-HCl (pH 8.3), 1.5 m
MMgCl2, 0.001% gelatin (final concentration) in a buffer solution of 20 nmol of deoxyribonucleotide triphosphate (dNTP: dATP, dCTP, dGT)
P, dTTP), a predetermined amount (here, 20 pmol each) of two types of primers (GH26, GH27; Ulf B. Gyllen
sten and Henry A. Erlich Proceeding National Academy of Sciences You. S. A (Proceeding National Academy of Science of US
A.) Vol.85, pp.7652-7656, October 1988) and 10 μl of genomic DNA extracted and purified by the above extraction method (1/10 of the extracted amount; about 50 to 100 ng), and the total amount is 1
After making up to 00 μl, heat-resistant DNA polymerase (Taq
Polymerase) is added. Finally, about 60 μl of mineral oil is added to prevent evaporation of the reaction solution. The primer amount is changed at the time of searching for the optimum condition, but this is changed by controlling the dispensing amount when dispensing each primer. The composition of the buffer solution and the amount of dNTP are not changed here, but the solution containing the salt to be controlled or dNTP is not changed.
It is also possible to change these values by controlling the dispensed amount of the solution.

The PCR reaction solution prepared as described above was attached to the container insertion hole 83 of the heating element 10 (PCR reaction tank), and the temperature of the reaction tank was changed to denature, anneal, DNA.
PCR is performed by cyclically changing each temperature of extension. Since it is possible to control the reached temperature for every four holes when searching for the optimum conditions, PCR can be performed simultaneously under four or more cycle number conditions under a maximum of four temperature conditions, and the results can be compared. . Further, it is possible to perform PCR up to 16 samples at the same time.

In the present Example, asymmetric PCR in which PCR is controlled asymmetrically to normal PCR (first step) and the primer amount ratio is controlled asymmetrically to specifically amplify only one side of the DNA double strand.
It is done in two stages (second stage). It is important to amplify the target single-sided single-stranded DNA by asymmetric PCR because the sequence reaction for determining the nucleotide sequence requires only one-sided single-stranded single-sided double-stranded DNA. In principle, it is much smaller than that of ordinary PCR, so it is necessary to amplify the target region in advance by ordinary PCR (Ulf B. Gyllensten and Henry A. Erlic
h Proceeding National Academy of Sciences You. S. A (Proceeding Nationa
l Academy of Science of USA) Vol.85, pp.7652-765
6, October 1988).

Then, the target double-stranded DNA amplified by the usual PCR as described above is introduced into the asymmetric PCR. That is, the same buffer solution as above (50 mM
KCl, 10 mM Tris-HCl (pH 8.3),
20 nmol of each deoxyribonucleotide triphosphate (dNTP: dATP, dCTP, dGTP, 1.5 mM MgCl2, 0.001% gelatin (final concentration))
The mixture of dTTP) was mixed with a predetermined amount ratio of two kinds of primers (in this case, GH26; excess amount, GH27 with M13 anchor primer sequence connected; limited amount), and the buffer solution at the first step PCR. 1 μl of the product of
Bring in 0 amount). Add Taq polymerase to 2.5
Unit mix and finally cover the top with mineral oil and close the lid and attach to a PCR reactor. After this, a predetermined number of cycles PC
By performing R, the second-stage asymmetric PCR is completed. In the second-stage asymmetric PCR, the primer amount ratio and P
The number of CR cycles is the main optimization parameter, but when searching for optimum conditions, it is possible to change these as in the case of the first-stage PCR and compare the results. In the present Example, the one in which the M13 anchor sequence was connected to the limiting amount side primer of the asymmetric PCR was used, but this is because the common anchor primer is used as the primer in the next sequence reaction.

P under various conditions for optimization condition search
FIG. 18 shows an example of the result of performing CR and performing analysis by gel electrophoresis using a simple electrophoresis apparatus. This graph is the result of optimization of the first-stage PCR with the human leukocyte antigen (HLA) gene DQα region as the target region, and the number of PCR cycles is plotted on the horizontal axis and plotted for each annealing temperature. As shown in FIG. 18, the amount of the main reaction product and the amount of the side reaction product continuously change according to the change of each parameter, and the increase rate of the amount of the main reaction product with respect to the number of PCR cycles is saturated and slowed down therein. There is a point. The cycle number immediately before the start of this blunting is the cycle number condition that maximizes the ratio of the amount of the main reaction product to the amount of the side reaction product, that is, the optimum cycle number (about 27 cycles in this case). Further, when the rate of increase of the amount of main reaction product with respect to the number of PCR cycles is compared for each annealing temperature, the rate of increase decreases at a certain annealing temperature. The temperature just before the start of the decrease of the increase rate is the optimum annealing temperature, which is 57 ° C. in this embodiment.

The PCR product obtained by completing the first-stage and second-stage asymmetric PCR under the optimum conditions is purified and concentrated by isopropanol precipitation as shown below. That is, mineral oil is removed from the reaction solution that has undergone the asymmetric PCR, and a 1 / 2M amount of a 5M ammonium acetate aqueous solution is added to adjust the final concentration to 1.5 to 2M. An equivalent amount of isopropanol is added thereto, and after mixing and stirring, the mixture is centrifuged at 10,000 × g for 30 minutes. Then, the supernatant is removed by decantation, and the pellet-like precipitate is subjected to the above-mentioned ethanol rinsing operation three times. This is necessary to remove the sodium salt used in the isopropanol precipitation, and lack of this desalting may hinder the next reaction, the sequence reaction. The sample after the ethanol rinsing operation is dried by a vacuum drying element as in the case described above, then dissolved in 7 μl of sterile deionized distilled water, and brought into a sequence reaction.

The process of the sequence reaction will be described.

The Taq sequencing reaction solution (buffer: 10 mM Tri) was added to the PCR product which had been purified as described above.
s-HCl (pH 8.5), 6 mM MgCl2, 1p
mol Fluorescent Label Sequencing Primer, 2unit
Add Taq polymerase to bring the total volume to 15 μl. Dispense 3.5 μl of this into 4 sample vessels. Then 4
Add the termination solution (mixed with 4 kinds of deoxyribonucleotide triphosphate and one kind of dideoxyribonucleotide triphosphate at a predetermined ratio) to each of the distributed containers, add mineral oil in an appropriate amount. , The heating element 10 (PCR reaction tank). After this, one cycle or more PCR temperature cycles are performed to complete the sequence reaction (Taq sequence or cycle sequence reaction). After the reaction,
Add 2 μl of reaction stop solution (95% formamide),
End the sequence reaction. Although the sequence reaction using Taq polymerase was described here, T7 was used.
A sequencing reaction using another enzyme such as DNA polymerase can be similarly realized.

The above is the pretreatment of all the gene analysis processes in this example. In this way, all the gene analysis processes such as extraction and purification of DNA from a biological sample, optimization and replication of reaction conditions for target DNA, replication amplification, purification of PCR amplification products, and nucleotide sequencing reaction should be carried out fully automatically. Is possible. By the way, if the optimum condition search process is not performed, the entire process can be completed in about 8 to 9 hours.

In particular, as is apparent from the heating and vacuum drying elements shown in FIG. 11, in the present invention, one sample can be reacted at a time under different conditions. Since the data as shown in FIG. 18 under the condition can be obtained at one time, the guideline for optimization can be easily obtained.

The above embodiment is only one embodiment of the present invention, and the present invention can be applied to various gene analysis processes which can realize each reaction element.

[0092]

According to the present invention, the annealing temperature, the denaturing temperature, the number of cycles, and the amount of primer were optimized as the parameters of PCR, and PCR direct sequencing was performed using the obtained optimum values. As a result, good DNA sequencing results were obtained. Can be obtained constantly.

That is, according to the present invention, DNA extraction from blood or other biological samples, purification, specific gene DN
It is possible to automatically perform a medically useful gene analysis process such as replication amplification of A and enzymatic reaction for detection without interruption, and always prepare a favorable pretreatment reaction product for gene analysis.

As a result, the throughput of analysis of medically useful genetic information is improved and the reliability of the analyzed genetic information is increased. As a result, it is considered to make a great contribution to the practical application of gene diagnosis.

[Brief description of drawings]

FIG. 1 is a block diagram showing a device configuration of the present invention.

FIG. 2 is a schematic perspective view of the device of the present invention.

FIG. 3 is a plan view showing a container transport device around a dispensing element according to the present invention.

FIG. 4 is a view showing a lid opening mechanism of the dispenser according to the present invention.

FIG. 5 is a view showing a lid closing mechanism of the pipetting machine according to the present invention.

FIG. 6 is a view showing a transfer dispenser of the dispenser according to the present invention.

FIG. 7 is a diagram showing a pressure-feeding dispenser of the dispenser according to the present invention.

FIG. 8 is a plan view showing a centrifugal separation element according to the present invention.

FIG. 9 is a diagram showing a centrifugal bucket of the centrifuge in the present invention.

FIG. 10 shows a mixing element according to the invention.

FIG. 11 is a diagram showing a heating and vacuum drying element according to the present invention.

FIG. 12 is a view showing a container inverting element according to the present invention.

FIG. 13 is a diagram showing a gripping mechanism portion of a carrying element according to the present invention.

FIG. 14 is a diagram showing a treatment of waste according to the present invention.

FIG. 15 is a diagram showing a simplified gel electrophoresis element according to the present invention.

FIG. 16 is a view showing a signal pattern detected when a PCR product is electrophoresed by the gel electrophoresis tank of the present invention as an example.

FIG. 17 shows the gene pretreatment process adopted in this example.

FIG. 18: PC under various conditions for optimization condition search
The figure which shows an example of the result of having performed R and analyzing by gel electrophoresis by a simple type | mold electrophoresis apparatus.

[Explanation of symbols]

1: Container, 2: Container carrier, 3: Lid opening mechanism, 4: Lid closing mechanism, 5: Chip, 6: Chip stand, 7: Transfer dispenser (pipetter), 8: Reagent bottle, 9: Freezing and Refrigerator, 1
0: warming and vacuum dryer, 11: centrifuge element, 1
2: Mixing element, 13: Container reversing element, 14: Container conveying element, 15: Transfer dispensing tip waste hole, 16: Waste liquid hole, 1
7: Device case, 21a: Pressure delivery dispenser, 84: Temperature control block, 87: Closed container, 130: UV (ultraviolet) transilluminator, 131: Gel electrophoresis element, 14
9: Device main body control unit, 150: Operator intervention terminal device, 151: Electrophoresis result analysis device, 152: Storage device, 153: Vacuum pump, 154: Dispensing element.

Claims (4)

[Claims] 1. A PC having a plurality of reaction sections which can be independently controlled.
An R reaction vessel is provided, and the reaction is performed under a plurality of different reaction conditions at the same time. Under each reaction condition, the reaction condition that maximizes the ratio of the main reaction product amount and the side reaction product amount is set as the optimum reaction condition. A gene analysis apparatus configured to execute a program for searching and determining an optimum reaction condition, and to perform PCR and biochemical reactions before and after PCR by using the optimum reaction condition determined by the reaction condition searching program. 2. The PCR reaction tank is divided into a plurality of temperature control blocks and a temperature control mechanism for simultaneously controlling the temperature of a reaction sample solution at a plurality of different temperatures. The gene analysis device according to claim 1. 3. A gel electrophoresis device as a device for detecting the reaction product, which comprises a gel and a gel electrophoresis device containing ethidium bromide in a running buffer, and a predetermined amount of the reaction product at a predetermined place on the gel. The gene analyzing apparatus according to claim 1, further comprising a reaction solution injection mechanism for injecting a solution. 4. As an algorithm for searching the optimum reaction conditions, the amount of the main reaction product and the amount of the side reaction product of PCR are calculated from the amount of the detected PCR product, and the increase rate of the main reaction product with respect to the number of cycles and the side reaction product. The maximum annealing rate and the optimal primer amount are the ones that have the largest ratio to the increase rate, and the number of cycles is one cycle less than the number of cycles based on the number of cycles at which the rate of increase of the main reaction product with respect to the number of cycles is saturated. The gene analysis device according to claim 1, wherein the cycle number is adopted and stored as the optimum cycle number.