WO2017159084A1 - Electrophoresis chip, electrophoresis device and electrophoresis system - Google Patents

Abstract

The electrophoresis chip 1 according to the present invention comprises an electrophoresis tube 100 housing a gel for electrophoresis therein, a positive electrode 300 applying a positive potential to the lower end side of the electrophoresis tube, a negative electrode 400 applying a negative potential to the upper end side of the electrophoresis tube 100, and a holder 200 integrally holding the electrophoresis tube 100 and the both electrodes 300 and 400.

Description

Electrophoresis chip, electrophoresis apparatus, and electrophoresis system

The present invention relates to an electrophoresis chip, an electrophoresis apparatus, and an electrophoresis system for separating or analyzing organic substances such as biological substances by electrophoresis.

In recent years, next-generation sequencers have been put into practical use by Illumina and Roche. Furthermore, the amount of base sequence determination for DNA and the like is dramatically increased by using single molecule real-time sequencing or the like. As described above, although the next-generation sequencer technology has progressed dramatically, the preparation (preparation process) of samples such as DNA used for sequence analysis requires a complicated and manual process.

As described in Non-Patent Document 1, the pretreatment step of a sample such as DNA includes (a) DNA extraction from the sample, (b) DNA fragmentation, (c) DNA size selection (sizing), (D) Smoothing of DNA ends, (e) Addition of adapter sequence to DNA ends, (f) Purification of DNA, (g) Amplification of DNA are required. For DNA size selection, a technique using electrophoresis is known. DNA size selection for next-generation sequencers requires DNA fragments that are aligned in a predetermined range of size, for example, DNA fragments that are sized to 10 kbp to 25 kbp.

As the electrophoresis in the above (C) sizing, for example, agarose gel electrophoresis is used. In agarose gel electrophoresis, a negatively charged DNA sample moves to the positive electrode side in an agarose gel by applying a voltage to an agarose gel arranged in a flat plate shape. During this movement, long DNA fragments move slowly through the network gel, while short DNA fragments move quickly through the network gel. DNA fragments are separated as a band for each size by utilizing such a difference in moving speed. And the part of the band of a required size is cut out manually from an agarose gel, and the process after said (d) is performed. Regarding electrophoresis of DNA fragments, a separation / sorting apparatus using capillary gel electrophoresis as shown in Patent Document 1 has been proposed by Kanbara et al.

JP 2002-48766 A

Conventional electrophoresis has been difficult to process a relatively large amount of samples such as DNA at a time. The apparatus described in Patent Document 1 has difficulty in processing a relatively large amount of samples at the same time. Accordingly, an object of the present invention is to provide a novel electrophoresis tool, electrophoresis device, and electrophoresis system that enable efficient sample sizing.

The present invention is configured as follows.

(Aspect 1) An electrophoresis tube containing an electrophoresis gel and a buffer, a first electrode for applying a positive or negative potential to the lower side of the electrophoresis tube, and a positive electrode on the upper side of the electrophoresis tube Or an electrophoretic device comprising a second electrode for applying a negative other potential, the electrophoretic device comprising the electrophoretic tube and a holder for integrally holding the first electrode. (Aspect 2) The electrophoresis tool according to Aspect 1, wherein the holder integrally holds the electrophoresis tube, the first electrode, and the second electrode. (Aspect 3) The electrophoresis tool according to Aspect 1 or 2, wherein the holder holds an upper part of the electrophoresis tube. (Aspect 4) In the electrophoretic device according to any one of Aspects 1 to 3, the holder includes a first electrode accommodating portion that accommodates the first electrode, and a first electrode that accommodates the second electrode. An electrophoretic device comprising two electrode housing portions.

(Aspect 5) The electrophoretic device according to Aspect 4, comprising an hinge portion connecting the first electrode housing portion and the second electrode housing portion. (Aspect 6) The electrophoresis tool according to Aspect 4, wherein the first electrode housing part and the second electrode housing part are separable. (Aspect 7) The electrophoretic device according to Aspect 6, wherein the first electrode housing portion has an opening into which the second electrode housing portion can be inserted. (Aspect 8) In the electrophoresis tool according to any one of aspects 1 to 7, the second electrode extends along the electrophoresis tube from the upper side to the lower side of the electrophoresis tube. Electrophoresis tool. (Aspect 9) The electrophoretic device according to Aspect 8, wherein the holder includes an extension extending to the lower side of the electrophoretic tube along the second electrode. (Aspect 10) The electrophoretic device according to aspect 9, wherein the extension portion includes a holding portion for holding the electrophoretic tube.

(Aspect 11) The electrophoretic device according to any one of aspects 1 to 10, wherein the holder includes an accommodation groove for accommodating the second electrode. (Aspect 12) The electrophoresis tool according to any one of aspects 1 to 11, wherein a lower end of the first electrode is inserted into the electrophoresis tube. (Aspect 13) The electrophoresis tool according to Aspect 1, wherein the holder includes a first fitting body integrally formed with the electrophoresis tube so as to be fitted with the first electrode. Ingredients. (Aspect 14) The electrophoresis device according to Aspect 15, wherein the holder includes a second fitting body integrally formed with the electrophoresis tube so as to be fitted with the second electrode. Ingredients. (Aspect 15) The electrophoresis device according to Aspect 13 or 14, wherein the holder is provided on an upper part of the electrophoresis tube.

(Aspect 16) The electrophoresis intelligent tool according to any one of aspects 1 to 14, wherein the electrophoresis tube includes a buffer storage tube that stores a buffer and a gel storage tube that stores the gel. Electrophoresis tool. (Aspect 17) The electrophoresis tool according to Aspect 16, wherein a lower end of the first electrode is disposed in the buffer housing tube. (Aspect 18) An electrophoretic device, the controller for controlling electrophoresis, the electrophoretic device according to any one of aspects 1 to 17, and the first electrode of the electrophoretic device A first power feeding unit, a second power feeding unit that feeds power to the second electrode of the electrophoresis tool, a dispenser, the second electrode of the electrophoresis tool, and a lower end of the electrophoresis tube. An electrophoresis apparatus comprising: a well to be inserted; and a moving unit capable of moving the electrophoresis tool and / or the dispenser three-dimensionally. (Aspect 19) In the electrophoresis apparatus according to Aspect 18, in order to perform pulse field gel electrophoresis, a power supply unit that periodically changes an electric field applied between the first electrode and the second electrode is provided. Electrophoresis device. (Aspect 20) The electrophoresis apparatus according to Aspect 19, wherein the power supply section periodically reverses the direction of the electric field.

(Aspect 21) The electrophoresis apparatus according to Aspect 18 or 19, wherein the power supply section periodically changes the intensity of the electric field. (Aspect 22) In the electrophoresis apparatus according to any one of Aspects 18 to 21, the moving unit moves the electrophoresis tool and / or the dispenser substantially linearly in the vertical direction. And a lateral movement unit that moves the electrophoresis device and / or the dispenser substantially linearly in the lateral direction. (Aspect 23) The electrophoresis apparatus according to Aspect 22, wherein the horizontal movement unit is mounted with the vertical movement unit and moves the vertical movement unit in a horizontal direction. (Aspect 24) In the electrophoresis apparatus according to any one of aspects 18 to 23, the well is a plurality of wells, and the moving unit moves the electrophoretic tool, thereby dividing the plurality of bands into the plurality of wells. Take the electrophoresis device. (Aspect 25) In the electrophoresis apparatus according to aspect 24, when the moving unit moves the electrophoretic tool, the control unit moves the electrophoretic tool above the well by the moving unit. Electrophoresis device.

(Aspect 26) In the electrophoretic device according to aspect 24 or 25, when the moving unit moves the electrophoretic tool, the control unit moves the first power supply unit and the second power supply unit from the first power supply unit. An electrophoretic device that stops feeding power to one electrode and the second electrode. (Aspect 27) The electrophoresis apparatus according to any one of aspects 18 to 26, wherein the well includes a plurality of containers arranged in an annular shape. (Aspect 28) The electrophoresis apparatus according to Aspect 27, wherein the well includes a buffer container surrounded by the plurality of containers. (Aspect 29) The electrophoresis apparatus according to Aspect 27 or 28, comprising a well rotation mechanism for rotating the well. (Aspect 30) The electrophoretic apparatus according to Aspect 29, wherein the well rotation mechanism switches the plurality of sorting units by rotating the well.

(Aspect 31) The electrophoresis apparatus according to Aspect 30, wherein when the well rotation mechanism rotates the well, the electrophoresis tool moves upward away from the well by the moving unit. (Aspect 32) In the electrophoretic device according to any one of aspects 29 to 31, when the well rotation mechanism rotates the well, the control unit controls the first power supply unit and the second power supply unit. An electrophoretic device that stops power supply from the unit to the first electrode and the second electrode. (Aspect 33) The electrophoresis apparatus according to Aspect 22 or 23, wherein the dispenser is connected to the longitudinal movement unit. (Aspect 34) In the electrophoretic device according to any one of aspects 22, 23, and 33, the longitudinal movement unit moves the first power feeding unit and the second power feeding unit integrally. Electrophoresis device. (Aspect 35) The electrophoresis apparatus according to any one of aspects 18 to 34, further comprising a detection device that detects a band or a ladder generated when a sample flows through the gel of the electrophoresis tube.

(Aspect 36) The electrophoresis device according to Aspect 35, wherein the detection device includes a detection end facing the electrophoresis tube of the electrophoresis tool, and a detection end that moves the detection end in a three-dimensional manner. An electrophoresis apparatus comprising a moving unit. (Aspect 37) The electrophoresis apparatus according to Aspect 36, wherein the detection end moving unit moves the detection end along the longitudinal direction of the electrophoresis tube. (Aspect 38) The electrophoresis apparatus according to Aspect 36 or 37, wherein the detection end moving unit moves the detection end so as to face a side surface of the electrophoresis tube or the well. (Aspect 39) In the electrophoresis device according to any one of aspects 36 to 38, the detection end moving unit is configured to perform the electrophoresis with the detection end facing the electrophoresis tube or the well. An electrophoresis apparatus that moves the detection end in a direction toward or away from a side surface of a tube or the well. (Aspect 40) The electrophoresis apparatus according to any one of aspects 36 to 37, wherein the detection end portion holds an irradiation end of an excitation light optical fiber and a light reception end of a fluorescence light receiving optical fiber.

(Aspect 41) The electrophoresis apparatus according to any one of aspects 36 to 40, wherein the detection end portion includes a curved surface along a contour of a side surface of the well. (Aspect 42) The electrophoresis apparatus according to any one of aspects 36 to 41, wherein the detection apparatus detects positions of a plurality of bands separated from a sample being electrophoresed. (Aspect 43) The electrophoresis apparatus according to any one of aspects 18 to 42, further comprising a nucleic acid extraction mechanism using magnetic particles. (Aspect 44) The electrophoresis apparatus according to any one of Aspects 18 to 43, comprising an amplification mechanism for amplifying a nucleic acid.

(Aspect 45) An electrophoresis system comprising a plurality of the electrophoresis apparatuses according to any one of aspects 18 to 44. (Aspect 46) In the electrophoresis system according to Aspect 45, the control unit detects the position of each band separated from the sample by using the detection device for each of the plurality of electrophoresis tools. And controlling the power supply to the second electrode and the first electrode of each of the plurality of electrophoretic devices based on the detection position of the band, thereby moving the bands in each electrophoretic device. Controlled electrophoresis system. (Aspect 47) In the electrophoresis system according to aspect 46, the control unit includes the first electrode and the second electrode so as to align the positions of the bands in each of the plurality of electrophoresis tools. Electrophoresis system that controls power supply to the electrodes. (Aspect 48) An electrophoresis method for performing electrophoresis of a sample using the electrophoresis apparatus according to any one of aspects 18 to 44. (Aspect 49) An electrophoresis method, wherein the electrophoresis system according to any one of aspects 44 to 47 is used to execute parallel processing of electrophoresis of a plurality of samples. (Aspect 50) The electrophoresis method according to Aspect 48 or 49, wherein the sample is a biological substance. (Aspect 51) The electrophoresis method according to any one of aspects 48 to 50, wherein the biological substance is DNA, RNA, or protein.

According to the electrophoresis tool, the electrophoresis apparatus, and the electrophoresis system of the present invention, it becomes possible to efficiently size, sort, or milk and analyze a sample.

1 is a perspective view of an electrophoresis chip according to a first embodiment of the present invention. It is a side view of the electrophoresis chip of FIG. It is a perspective view of the electrophoresis tube of FIG. It is sectional drawing in the longitudinal direction of the electrophoresis tube of FIG. FIG. 2 is a developed perspective view of a holder for the electrophoresis tube of FIG. 1. It is a top view of the holder of FIG. FIG. 6 is a developed side view of the holder of FIG. 5. It is an enlarged side view in the middle of mounting | wearing with an electrophoresis tube in the holder of FIG. It is a side view of the state which attached the electrophoresis tube to the holder of FIG. FIG. 7 is an enlarged perspective view showing that the holder is closed after the electrophoresis tube is attached to the holder of FIG. 6. FIG. 3 is a top view of the electrophoresis chip in FIG. 2. 1 is a side view of an electrophoresis apparatus according to a first embodiment of the present invention. It is a side view which shows the dispensing state which used the dispenser to the electrophoresis chip of FIG. It is a sectional side view which shows the electrophoresis using the electrophoresis apparatus of FIG. 1 is a top view of an electrophoresis system according to a first embodiment of the present invention. It is sectional drawing which shows the electrophoresis by the electrophoresis system of FIG. It is the (a) side view and (b) front view of the electrophoresis chip which concern on the 2nd Embodiment of this invention. It is the (a) side view and (b) front view of the positive electrode accommodating part of FIG. It is the (a) side view and (b) front view of the state which mounted | worn with the electrophoresis chip in the plus electrode accommodating part of FIG. It is a side view just before mounting | wearing the plus electrode accommodating part of Fig.19 (a) to a minus electrode accommodating part. It is a perspective view of the state of FIG. FIG. 18 is a top view of the electrophoresis chip in FIG. 17. It is a perspective view of the electrophoresis system which concerns on the 2nd Embodiment of this invention. It is a front view of the electrophoresis system of FIG. It is a side view of the electrophoresis system of FIG. FIG. 24A is a plan view of a four-lane arrangement and FIG. 24B is a plan view of an eight-lane arrangement regarding the stage of the electrophoresis system of FIG. FIG. 24 is a perspective view of a rotating sorting well used in the electrophoresis system of FIG. 23. It is a side view of the use condition of the rotation sorting well of FIG. It is a perspective view of the rotation mechanism of the rotation fractionation well of FIG. It is a principal part perspective view of the electrophoresis state of the electrophoresis system of FIG. It is a principal part perspective view of the detection end part moving unit of FIG. It is a principal part perspective view of the movement state of the detection end part moving unit of FIG. It is a side view of the various containers used for the Example of this invention. It is a figure which shows the electrophoresis method in the Example of this invention. It is a figure which shows the electrophoresis pattern in the Example of this invention. It is the (a) front view and the (b) side view of the electrophoresis chip concerning a 3rd embodiment of the present invention. FIG. 37 is a cross-sectional view of the upper part of the electrophoresis chip of FIG. 36. FIG. 37 is a side view showing a first assembly process of the electrophoresis chip in FIG. 36. FIG. 37 is a side view showing a second assembly step of the electrophoresis chip in FIG. 36. FIG. 37 is a side view showing a third assembly step of the electrophoresis chip in FIG. 36.

Embodiments of an electrophoresis chip (electrophoresis tool), an electrophoresis apparatus, and an electrophoresis system according to the present invention will be described. The electrophoresis chip of this embodiment is preferably an integrated electrophoresis tube, plus electrode, and minus electrode. The electrophoresis device of the present embodiment enables the electrophoresis chip to be moved three-dimensionally by attaching the electrophoresis chip to the moving unit. The electrophoresis system of the present embodiment includes a plurality of processing lanes (processing lines) of the electrophoresis apparatus, and processes a plurality of samples simultaneously in parallel. In each embodiment, the same parts are denoted by the same reference numerals. The description of the same part is omitted as appropriate.

[First Embodiment]
(Electrophoresis chip)
An electrophoresis chip (Tip) 1 according to a first embodiment of the present invention will be described with reference to FIGS. The electrophoresis chip 1 includes an electrophoresis tube 100 that accommodates an electrophoresis gel, a holder 200 that holds the upper part of the electrophoresis tube 100, and a positive potential (first potential) applied to the gel from below the electrophoresis tube 100. And a negative electrode (second electrode) 400 for applying a negative potential (second potential) to the gel from above the electrophoresis tube 100. The holder 200 fixes and integrates the electrophoresis tube 100, the plus electrode 300, and the minus electrode 400. Note that the positive electrode 400 may not be integrated by the holder 400, and the positive electrode 400 may be movably provided on the sorting wells 511 to 513 (cartridge) side in FIG.

3 and 4 show the state of the electrophoresis tube 100 before the start of use (before fixing to the holder 200). The electrophoresis tube 100 is filled in the upper end 108 held by the holder 200, a buffer storage tube 110 for storing a buffer, a gel storage tube 112 formed narrower than the buffer storage tube 110, and the gel storage tube 112. Gel 140. The electrophoresis tube 1 has a straw shape, and the inner diameter of the gel containing tube 112 can be preferably 3 mm to 4 mm. The electrophoresis tube 100 preferably includes a flexible upper cap 120 that closes the upper end opening of the electrophoresis tube 100 and a flexible lower cap 130 that closes the lower end opening. By providing both the caps 120 and 130 in the electrophoresis tube 100, before the electrophoresis tube 100 is attached to the holder 200, drying of the gel and contamination to the gel can be prevented. The upper cap 120 and the lower cap 130 are removed before performing electrophoresis.

Instead of the upper cap 120, an upper cap 121 for closing the upper portion of the gel 140 inside the electrophoresis tube 100 may be provided. The upper cap 121 is rod-shaped and includes a knob 123 that extends to the outside of the upper end opening of the electrophoresis tube 100. The upper cap 121 can be easily removed by pulling the knob 123 during use. Further, a ring may be attached to the end of the knob 123 so that the cap 123 can be easily pulled out. The electrophoresis tube 1 is made of a transparent material that can transmit ultraviolet light and visible light. Prior to electrophoresis, the buffer is dispensed using a dispenser 610 as shown in FIG. The gel 140 is preferably, but not limited to, an agarose gel or a polyacrylamide gel.

As shown in the developed views of FIGS. 5 and 6, the holder 200 includes a positive electrode housing portion (first electrode housing portion) 210 that houses the positive electrode 300 and a negative electrode housing portion (first electrode housing portion that houses the negative electrode 400. 2 electrode housing part) 220 and a hinge part 230 connecting both electrode housing parts 210 and 220. Each of the electrode housing portions 210 and 220 has a curved shape (a shape obtained by cutting the tube in the longitudinal direction) so as to surround and accommodate the upper portion of the electrophoresis tube 100. When the negative electrode housing part 220 is rotated with respect to the positive electrode housing part 210 around the hinge part 230, the two electrode housing parts 210 and 220 form a cylindrical holder 200. Although not shown, the holder 200 preferably includes a locking structure that fixes the electrode housing portions 210 and 220 to each other in a state where the cylindrical holder 200 is formed. The locking structure includes, for example, an elastically deformable claw provided in one electrode housing portion and a recess provided in the other electrode housing portion and hooked by the claw.

The plus electrode housing part 210 includes an electrode housing groove 211 for housing the plus electrode 300 and an extension part 213 extending to the vicinity of the lower end of the plus electrode 300. The electrode housing groove 211 extends from the upper end of the plus electrode housing portion 210 to the lower end of the extension portion 213. The extension 213 is preferably provided with a plurality of protrusions protruding on the electrode housing groove 211, so that the electrode can be prevented from falling off. Further, as shown in FIG. 1, the extension portion 213 holds one or more holding portions 215 that hold the gel storage tube 112 of the electrophoresis tube 100 and / or the lower end 320 side of the plus electrode 300. It is also possible to provide an electrode holding portion 217 to be used. The negative electrode housing part 220 includes a C-shaped protrusion 221 that detachably holds the negative electrode 400.

A state in which the plus electrode 300 and the minus electrode 400 are attached to the holder 200 of FIGS. 5 and 6 will be described with reference to FIG. The positive electrode 300 includes an upper end 310 connected to the positive power supply unit (first power supply unit) 621 in FIG. 12, a lower end 320 immersed in a buffer filled in the sorting wells 511 to 513 in FIG. 310 and an electric wire part 330 connecting the lower end 320. The electric wire part 330 is accommodated in the electrode accommodation groove 211.

The negative electrode 400 connects the upper end 410 connected to the negative power supply unit (second power supply unit) 623 in FIG. 12, the lower end 420 immersed in the buffer filled in the electrophoresis tube 100, and the upper end 410 and the lower end 420. The electric wire part 430 is configured. The minus electrode 400 is detachably held by a C-shaped protrusion 221 provided in the minus electrode housing part 220.

The procedure for mounting the electrophoresis tube 100 on the holder 200 in the state of FIG. 7 will be described. First, the caps 120 and 130 are removed from the electrophoresis tube 100 in the state shown in FIG. Next, as shown in FIG. 8, the electrophoresis tube 100 is moved from the lower side to the upper side of the negative electrode housing part 220, and the lower end 420 of the negative electrode 400 is introduced into the electrophoresis tube 100. Further, as shown in FIG. 9, the electrophoresis tube 100 is moved until the lower end 420 extends into the buffer storage tube 110. 9 to 10, the electrode housing portions 210 and 220 are closed, so that the holder 200 surrounds and fixes the upper part of the electrophoresis tube 100. This state is shown in FIGS. FIG. 11 is a view of the electrophoresis chip 1 in the state of FIGS. 1 and 2 as viewed from above.

(Electrophoresis device)
An electrophoresis device 2 according to a first embodiment of the present invention will be described. The electrophoresis device 2 includes an electrophoresis chip 1. As shown in FIG. 12, the electrophoresis apparatus 2 includes a vertical movement unit 600 that can move linearly in the vertical direction (substantially vertical direction) and a plurality of wells arranged in a substantially linear shape (line shape). And a line (cartridge) 500. In FIG. 12, illustration of the electrophoresis chip 1 is omitted. In the vertical movement unit 600, a dispenser 610 to which the dispensing tip 611 is detachably attached, a dispenser attachment portion (aspiration nozzle) 613 to which the dispenser 610 is detachably attached, and the electrophoresis chip 1 are provided. And an electrophoresis chip mounting portion 620 that is detachably mounted. The electrophoretic apparatus 2 includes a lateral movement unit (for example, the lateral movement unit 900 in FIG. 23) that allows the vertical movement unit 600 to move substantially linearly in the horizontal direction (substantially horizontal direction), the horizontal movement unit 600, and the vertical movement unit 600. A control unit (not shown) for controlling the moving unit 900. Since the electrophoresis apparatus 2 includes the vertical movement unit 600 and the horizontal movement unit 900, the electrophoresis chip 1 can be moved three-dimensionally on the processing line 600. The electrophoresis chip 1 is housed in an electrophoresis chip housing rack 520 shown in FIG. As shown in FIG. 13, the dispenser 610 can dispense a buffer or a sample from the upper end opening of the electrophoresis chip 1 into the inside thereof. Further, the dispenser 610 can dispense (discharge) a solution, a sample, and a reagent into each well, or can suck and move these from each well.

FIG. 14 shows a state in which electrophoresis is performed using the electrophoresis apparatus 2 of the first embodiment. The electrophoresis device 2 includes a detection device (fluorescence scanner) 700 that detects a band generated in the gel 140 by electrophoresis of a sample such as DNA. The detection device 700 includes an excitation light source 705, an excitation light optical fiber 710, a fluorescence light receiving optical fiber 720, a detection end 730 that holds an irradiation end of the excitation light optical fiber 710 and a light reception end of the fluorescence receiving optical fiber 720. A photodetection element 740 such as a photomultiplier tube (PMT) that detects light from the fluorescence receiving optical fiber 720, and a detection end moving unit that moves the detection end 730 three-dimensionally (for example, FIG. 32 detection end moving units 750). The detection end 730 is configured so that the irradiation end of the excitation light optical fiber 710 and the light reception end of the fluorescence light receiving optical fiber 720 are moved to the side surface of the gel containing tube 112 and / or the side surface of the well 510 by the detection end moving mechanism. Can be moved in the longitudinal direction (substantially vertical direction) along the longitudinal direction of the gel containing tube 112 and / or the well 510. Further, the detection end portion 730 can be moved in a lateral direction (substantially horizontal direction) approaching or leaving the side surface of the gel containing tube 112 and / or the side surface of the well 510 by the detection end portion moving unit. The sorting wells 511 to 513 are made of a transparent material that can transmit ultraviolet light and visible light.

The parallel processing type electrophoresis system 3 according to the first embodiment includes a plurality of electrophoresis apparatuses 2 to 2G having the same configuration as shown in FIG. As shown in the top view of the stage 800 in FIG. 15, each electrophoresis apparatus 2 to 2G includes an electrophoresis chip 1 to 1A, a processing line (processing lane) 500 to 500G, a vertical movement unit 600 to 600G, and a detection unit. A device 700 to 700G (not shown) and a lateral movement unit 900 are included. Each of the vertical movement units 600 to 600G can move along their longitudinal direction above the corresponding processing line 500 to 500G. Further, a detection end 730 of the detection device is provided for each of the processing lines 500 to 500G. Each detection end 730 is preferably arranged in the lateral movement direction of each electrophoresis chip 1 mounted on the vertical movement unit 600. As a result, as shown in FIG. 15, even when a plurality of electrophoresis apparatuses are provided, the horizontal movement of one detection end 730 does not interfere with the movement of the other detection end 730, and the entire arrangement is compact. can do. The sorting wells 511, 512, and 513 can be moved when sorting other wells so as not to interfere with the horizontal and vertical movement of the detection end 730. In FIG. 15, by providing individual horizontal movement units 600 to 600G for each vertical movement unit, it is possible to move three-dimensionally independently on each processing lane 500 to 500G, but each vertical movement unit is integrated. A vertical movement unit (vertical movement unit 600 in FIG. 23) and / or a horizontal movement unit (lateral movement unit 900 in FIG. 23) may be provided.

(Preparation by electrophoresis)
The process of fractionating DNA (sample) by electrophoresis using the electrophoresis apparatus 2 will be described with reference to FIG. DNA is extracted from the sample using a known technique and fragmented. A plurality of labeled DNAs (molecular weight markers) are added to the obtained fragmented DNA. Each of the plurality of labeled DNAs has a known molecular weight (size) and can be detected using the detection device 700 by fluorescence. For example, two types of labeled DNA having a molecular weight of 10 kbp and labeled DNA having a molecular weight of 25 kbp can be added. When electrophoresis is carried out with a plurality of labeled DNAs added in this way, a plurality of labeled DNA bands and a fragmented DNA band located between each labeled DNA band are separated. Then, by detecting the position of the band of the labeled DNA with the detection device 700, the fragmented DNA having a required molecular weight can be sorted (milked).

The specific processing procedure of sorting (milking) by the electrophoresis apparatus 2 is as follows. The electrophoresis chip 1 assembled in the state of FIGS. 1 and 2 is set in the chip storage rack 520 (FIG. 15) of the electrophoresis apparatus 2. The electrophoresis chip 1 is not attached to the longitudinal movement unit 600 while being accommodated in the chip accommodation rack 520. In this state, as shown in FIG. 13, the controller uses a dispenser 610 to perform electrophoresis of a buffer, a fragmented DNA sample, and a plurality of labeled DNAs of known molecular weight from the upper end opening of the electrophoresis chip 1. Dispense into the tip 1. Next, the upper part of the electrophoresis chip 1 is mounted on the electrophoresis chip mounting part 620 of the vertical movement unit 600. Simultaneously with this mounting, the upper end 310 of the positive electrode 300 is inserted into the positive power feeding unit 621 of the vertical movement unit 600 and the upper end 410 of the negative electrode 400 is inserted into the negative power feeding unit 623 of the vertical movement unit 600 and electrically connected. To do. As a result, a voltage can be applied to the gel 140 (FIG. 4). Note that the electrophoresis chip mounting portion 620 may be provided with a pressing mechanism that holds and holds the upper part of the electrophoresis chip 1 in order to securely fix the electrophoresis chip 1 when the electrophoresis chip 1 is moved.

When a voltage is applied to the gel 140, the DNA is negatively charged and moves toward the lower end 320 of the positive electrode. During this movement, the larger the size (molecular weight) of the DNA, the slower the movement speed in the gel, so that the DNA is separated into bands A, B, and C as shown in FIG. In this embodiment, band A is a 25 kbp labeled DNA, band B is a fragmented DNA that needs to be sorted, and band C is a 10 kbp labeled DNA.

While the detection end portion 730 of the detection device 700 is opposed to the gel storage tube 112, the gel storage tube 112 is irradiated with excitation light while moving in the longitudinal direction of the gel storage tube 112, and the fluorescence from the gel storage tube 112. Can be measured. Thereby, the separation state of the fragmented DNA in the longitudinal direction of the gel containing tube 112 can be confirmed in real time. The detection end portion 730 is movable in the vertical direction and the horizontal direction between the upper end and the lower end of the gel containing tube 112.

The detection device 700 indicates that the band C (labeled DNA) has fallen into the sorting well 511 with the detection end 730 facing the side surface of the lower end of the electrophoresis tube 100 and the side wall of the sorting well 511. Can be confirmed. After the control unit confirms that the sorting of the band C is completed by a signal from the detection device 700, the control unit stops applying the voltage from the power feeding units 621 and 623 to the electrodes 300 and 400, and the other band A And B (fragmented DNA) stop moving through the gel. With the voltage application stopped, the lower end of the electrophoresis chip 1 is moved into the well 512 using the longitudinal movement unit 600. When the lower ends of the electrophoresis chip 1 and the plus electrode 300 are immersed in the buffer of the well 512, the control unit resumes voltage application from the power supply units 621 and 623 to the electrodes 300 and 400. With the resumption of voltage application, band B starts moving downward. Preferably, the lower ends of the electrophoresis chip 1 and the electrode 300 can be washed before moving to a different well.

After the control unit of the electrophoresis apparatus 2 or the electrophoresis system 3 according to the first embodiment confirms that the fragmented DNA of the band B has been dropped into the well 512 and can be sorted by a signal from the detection apparatus 700, The control unit again stops voltage application from the power supply units 621 and 623 to the electrodes 300 and 400 to stop the band A from moving in the gel. In this way, fragmented DNA and labeled DNA contained in a plurality of bands can be separated (milked) while moving the electrophoresis chip 1.

A case will be described in which a plurality of electrophoresis chips 1 to 1G are provided and electrophoresis of the same sample is performed simultaneously as in the parallel processing type electrophoresis system 3 of FIG. Even if the same sample is supplied to the gels accommodated in the electrophoresis chips 1 to 1G and the electrophoresis is performed simultaneously, the samples cannot be completely separated at the same band position. For example, as shown in FIG. 16, the positions of bands A to C are different in the electrophoresis chips 1 to 1B. This is because the gel state cannot be made completely uniform. It is difficult to adjust such a deviation in normal electrophoresis.

In the electrophoresis system 3, the position of each band is measured by the detection end portion 730 provided in each of the electrophoresis chips 1 to 1B, and the control unit adjusts the voltage supply, thereby controlling the lowering of each band. The position (height) can be aligned. Thereby, the band which needs to be sorted can be sorted into each sorting well 511 at the same timing. For example, the control unit stops supplying voltage to the electrophoresis chip 1 having the necessary band position below, while the control unit supplies voltage to the electrophoresis chip 1 having the necessary band position above. By continuing, the position of each band can be adjusted to the same height.

A fluorescent scanner suitable for the detection apparatus 700 of the electrophoresis system 3 has been filed as a patent application No. 2015-94426 by the applicant of the present application (invention name “Multiple reaction parallel measurement apparatus and method”). The case where the fluorescent scanner described in the patent application is applied to the present invention will be described. Each light irradiation detection unit 730 provided in each electrophoresis chip is attached to a common vertical movement frame extending in the horizontal direction. The light emission ends of the fluorescence from each fluorescence receiving optical fiber 720 of each light irradiation detection unit 730 are arranged concentrically, and a rotating rotary fiber rotating unit 750 (FIGS. 23 and 25) is provided for this arrangement. The fluorescence is sequentially incident on one optical fiber provided on the rotating body. Thus, the fluorescence of the plurality of electrophoresis chips can be guided to one optical fiber, and the fluorescence of the plurality of electrophoresis chips can be detected and processed in a time-sharing manner by the light detection element connected to the one optical fiber.

[Second Embodiment]
(Electrophoresis chip)
An electrophoresis chip 1 ′ according to a second embodiment of the present invention will be described. The electrophoresis chip 1 ′ (FIG. 17) is different from the electrophoresis chip 1 in the structure of the holder 1200. The holder 1200 includes a plus electrode housing portion 1210 (FIG. 18) that houses the plus electrode 300 and a minus electrode housing portion 1220 (FIG. 20) that houses the minus electrode 400. The plus electrode housing portion 1210 and the minus electrode housing portion 1220 are separable members.

As shown in FIG. 18, the plus electrode housing portion 1210 has a curved shape (a shape obtained by cutting the tube in the longitudinal direction) so as to surround and accommodate the upper portion of the electrophoresis tube 100. The positive electrode housing portion 1210 includes an extension portion 213 similar to that of the first embodiment. When the electrophoresis tube 100 is attached to the positive electrode housing portion 1210, the state shown in FIG. As shown in FIGS. 18 and 19, the negative electrode housing portion 1220 is substantially cylindrical and includes a flange portion 1223 provided at the lower end and a positioning rib 1225 protruding downward from the flange portion 1223. By providing a slit into which the positioning rib 1225 can be inserted in the accommodation opening of the stage 800 or the electrophoresis chip mounting part 620 of the longitudinal movement unit 600, the electrophoresis chip 1 'can be held in an appropriate orientation.

The upper part of the plus electrode accommodating part 1210 equipped with the electrophoresis tube 100 is moved in the direction of the arrow in FIGS. 20 and 21 and inserted into the opening of the minus electrode accommodating part 1220. As a result, the electrophoresis tube 100, the plus electrode housing portion 1210, and the minus electrode housing portion 1220 are integrated to complete the assembly of the electrophoresis chip 1 '. As shown in FIG. 22, the assembled electrophoresis chip 1 ′ can be dispensed into the electrophoresis tube 100 through its upper end opening. The electrophoresis chip 1 ′ may include a drop-off prevention mechanism that prevents the positive electrode storage portion 1210 from dropping off from the negative electrode storage portion 1220. The drop-off prevention mechanism includes a rib or projection provided inside the cylindrical portion of the negative electrode housing portion 1220 and a recess provided on the outer surface of the negative electrode housing portion 1220 with which the rib or projection meshes. The ribs or protrusions engage with the recesses to prevent falling off.

(Electrophoresis system)
An electrophoresis system 3 according to a second embodiment of the present invention will be described. The electrophoresis system 3 embodies the electrophoresis system 3 according to the first embodiment. As shown in FIGS. 23 to 25, the electrophoresis system 3 includes a detection mechanism (fluorescence scanner) 700, a stage 800 including a plurality of wells including wells 530 and the like, and a vertical direction (substantially approximately) provided on the stage 800. A vertical movement unit 600 that can move in a substantially straight direction (vertical direction), and a horizontal movement unit 900 that can move in a substantially straight direction in the horizontal direction (substantially horizontal direction) with the vertical movement unit 600 mounted. . The vertical movement unit 600 is detachably mounted with the electrophoresis chip 1 or 1 ′. The detection mechanism 700 includes a rotary fiber rotation mechanism 750.

The horizontal movement unit 900 includes a motor 901 that moves the vertical movement unit 600 in the vertical direction and a motor 903 that drives a pump that performs suction / discharge of the dispenser 601 of the vertical movement unit 600. Since the vertical movement unit 600 and the motors 901 and 903 are mounted on the horizontal movement unit 900, the vertical movement unit 600 and the motors 901 and 903 can move in the horizontal direction integrally with the horizontal movement unit 900. The lateral movement unit 900 includes a lateral movement mechanism (motor and gear) (not shown) under the stage 800.

The structure of the stage 800 will be described with reference to FIG. FIG. 26A shows a four-lane arrangement in a state in which the electrophoresis sorting well 530 is mounted on the stage 800, and FIG. 26B shows that the sorting well 530 is removed from the stage 800 and non-electrophoresis non-electrophoresis. This is an 8-lane arrangement with the sorting well 535 attached. The stage 800 includes a plurality of processing lines (processing lanes) in which a plurality of wells and the like are arranged in a line. FIG. 26 (b) shows an arrangement using eight processing lines. FIG. 26 (a) uses a rotary sorting well 530 having a diameter larger than the line width, so four processing lines are skipped for one lane. Is an arrangement that uses The arrangement shown in FIG. 26B is used when it is not necessary to take a sample and information on the positional relationship (ladder shape) of the band is necessary (inspecting the parent-child relationship).

Each line of the stage 800 includes a sample tube 580, a chip storage cartridge 550, a sample extraction cartridge 560, a PCR cartridge 570, a sorting well 530 (FIG. 26 (a)), or a non-sorting well 535 (FIG. 26 ( b)). The sample tube 580 contains a sample of the biological material. In the chip storage cartridge 550, a plurality of dispensing chips 611 (FIG. 12), a piercing chip for perforating the seal of the reagent container, and the electrophoresis chip 1 or 1 'are stored. The sample extraction cartridge 560 includes a plurality of wells, and extracts biological materials such as DNA from the sample using magnetic particles. Further, as shown in FIG. 26, the horizontal movement unit 900 moves integrally with the vertical movement unit 600 along the longitudinal direction of the processing line above the stage 800. In the vertical movement unit 600, the dispenser 610 or the electrophoresis chip 1 (1 ′) attached to the vertical movement unit 600 is moved above the necessary wells on the processing line by the horizontal movement unit 900, It descends by the vertical movement unit 600 and executes a predetermined process. The predetermined processing can be piercing by the dispenser 610, suction, or ejection, or sorting by electrophoresis using the electrophoresis chips 1 and 1 '.

(Preparation well)
The sorting well 530 and the sorting well rotating mechanism 540 used in the second embodiment of the present invention will be described with reference to FIGS. The sorting well 530 is formed in a substantially bottomed cylindrical shape using a transparent material that can transmit ultraviolet light and visible light. Further, the sorting well 530 includes a buffer container 531 for accommodating a buffer for electrophoresis and the like, a plurality of sorting containers 533 provided in an annular shape so as to surround the buffer container 531, and a cross shape from the bottom of the buffer container 531. And a protruding portion 535 that protrudes from the center. At the time of electrophoresis, as shown in FIG. 28, a buffer previously stored in the buffer container 531 is dispensed into the sorting container 533 using a dispenser 610. The plurality of sorting containers 533 can include a sorting container 533 for sample sorting and a sorting container 533 for drainage sorting.

The sorting well rotation mechanism 540 rotates the plurality of electrophoresis sorting wells 530 using a rack and pinion. As shown in FIG. 29, the sorting well rotating mechanism 540 includes a sorting well rotating motor 541, a first pinion gear 543 that is rotated by the motor 541, and a rack gear 545 that is linearly moved by the first pinion gear 543. The plurality of second pinion gears 547 rotated by the rack gear 545 and the plurality of internal gears 549 rotated by the plurality of second pinion gears 547 are provided. The rack gear 545 includes a first tooth surface 543a that meshes with the first pinion gear 543, and a second tooth surface 543b that meshes with the plurality of second pinion gears 547. The internal gear 549 is fixed to the protruding portion 535 of the sorting well 530. When the control unit changes the rotation direction of the motor 541, the plurality of electrophoresis sorting wells 530 can be rotated clockwise or counterclockwise. By the sorting well 530 and the sorting well rotating mechanism 540, a plurality of bands in the sample can be sorted into a plurality of sorting containers 533 without moving the position of the lateral movement unit 900.

The operation of sorting DNA or the like by the electrophoresis apparatus 3 according to the second embodiment will be described with reference to FIGS. FIG. 30 shows a state where electrophoresis is performed with the electrophoresis chip 1 ′ attached to the electrophoresis apparatus 3. The lower end of the electrophoresis tube 100 and the lower end 320 of the positive electrode 300 of the electrophoresis chip 1 ′ are immersed in the buffer of the sorting container 533 of the sorting well 530. The detection end 730 of the detection device 700 is disposed behind each sorting well 530. The detection end part (fluorescence scanning part) 730 can be moved substantially linearly in the vertical direction (substantially vertical direction) and the horizontal direction (substantially horizontal direction) indicated by arrows in FIG. 31 by the detection end part moving unit 750. . The detection end 730 includes a curved surface 731 that is curved so as to have the contour of the side surface of the sorting well 530, and a slit 731 a provided on the curved surface 731. Inside the slit 731 a, the irradiation end of the excitation light optical fiber 710 and the light receiving end of the fluorescence light receiving optical fiber 720 are disposed so as to face the side surfaces of the sorting well 530 or the gel storage tube 112 of the electrophoresis tube 100. Yes. The detection end moving unit 750 includes a plurality of detection end portions 730, a vertical movement mechanism (not shown) that moves the plurality of detection end portions 730 integrally or individually in a substantially vertical direction, and a plurality of detection end portions. A lateral movement mechanism (not shown) that moves 730 integrally or individually in a substantially lateral direction.

In the electrophoresis, the detection end 730 is movable as shown in FIG. 32 along the longitudinal direction (substantially vertical direction) of the electrophoresis chip 1 ′. When the detection end 730 moves along the longitudinal direction, the detection end 730 can move in a direction approaching or moving away from the side surface of the electrophoresis chip 1 ′. Accordingly, the measurement accuracy is improved because the band can be measured in a state where the detection end portion 730 is opposed in the vicinity of the side surface of the electrophoresis chip 1 ′. As in the case described with reference to FIG. 14, by performing measurement while moving the detection end 730 in a substantially vertical direction, the positions of a plurality of bands separated from the sample during electrophoresis can be scanned.

In the state where the detection end 730 is at the position shown in FIG. 30, the detection end 730 can measure the lowermost end of the electrophoresis chip 1 ′. When it is detected that one band has fallen on one sorting unit 533, the control unit of the electrophoresis system 3 stops applying voltage to the plus and minus electrodes 300 and 400, and then the lower end of the electrophoresis chip 1 ′. Is moved above the sorting well, the sorting well 530 is rotated by a predetermined angle, and the electrophoresis chip 1 ′ is moved above the other sorting section 533. Then, the control unit lowers the electrophoresis chip 1 ′, immerses the lower end of the electrophoresis chip 1 ′ in a buffer in another sorting unit 533, and applies a voltage between the plus and minus electrodes 300 and 400. Resume electrophoresis. By repeating such an operation, a plurality of bands can be automatically sorted into a plurality of sorting units.

[Third Embodiment]
The electrophoresis chip 1 of the first embodiment (FIGS. 1, 14, and 16), the electrophoresis chip 1 ′ of the second embodiment (FIG. 17), and the electrophoresis chip 1A of the fourth embodiment (FIG. 36). ), A positive potential is applied to the gel from the lower side of the electrophoresis tube 100 by the first electrode (plus electrode) 300, and a negative potential is applied to the gel from the upper side of the electrophoresis tube 100 by the second electrode (negative electrode) 400. ing. As a result, when electrophoresis is performed using the electrophoresis chips 1, 1 ′, 1 A, samples such as DNA are separated according to the molecular weight while being moved from top to bottom and can be detected or sorted.

On the other hand, in the third embodiment, the first electrodes 300 and 300A on the lower side of the first to fourth embodiments are set as negative electrodes, and the second electrodes on the upper side of the first to fourth embodiments are used. The electrodes 400 and 400A were positive electrodes. Furthermore, in the third embodiment, the first power feeding unit 621 in FIG. 12 is a negative electrode, and the second power feeding unit 623 is a positive power feeding unit. When electrophoresis is performed using the electrophoresis chip 1, 1 ′ of the third embodiment, a sample such as DNA is provided from the lower end side of the electrophoresis tube 100 of the electrophoresis chip 1, 1 ′. The sample is separated according to the molecular weight while moving from bottom to top, and can be fractionated. It should be noted that by adjusting the magnitude of the voltage applied to the top and bottom of the electrophoresis tube 100 of the third embodiment, the sample can be electrophoresed from bottom to top regardless of gravity.

[Fourth Embodiment]
Unlike the electrophoresis chip 1 of the first embodiment and the electrophoresis chip 1 ′ of the second embodiment, the electrophoresis chip 1A of the fourth embodiment is different from the first electrode (plus electrode) 300A and the second electrode. A holder for holding an electrode (minus electrode) 400 </ b> A is integrally formed with the electrophoresis tube 100. An electrophoresis chip 1A according to a fourth embodiment will be described with reference to FIGS. The electrophoresis tube 100 of the electrophoresis chip 1 </ b> A includes a buffer housing tube 110 and a gel housing tube 112 (capillary tube) that is press-fitted into the lower end of the buffer housing tube 110. The gel containing tube 112 is filled with a gel 140, and the lower end opening of the gel containing tube 112 is sealed with a lower cap (rubber plug) 130. The buffer housing tube 110 protrudes upward from the flange portion 130, a plurality of ribs 131 formed between the flange portion 130 and the side surface of the buffer housing tube 110, and a flange portion 130 formed around the upper end opening. A first electrode fitting pin (first fitting body) 133 and a second electrode fitting pin (second fitting body) 134 protruding upward from the flange portion 130 are provided. The holder according to the fourth embodiment includes a first electrode fitting pin 133 and a second electrode fitting pin 134.

A notch 133a is provided at the upper end of the first electrode fitting pin 133, and the curved portion of the first electrode 300A is accommodated or fitted in the notch 133a. A pair of through holes are provided in the flange portion 130 around the lower end of the first electrode fitting pin 133. The upper end 310A and the lower end 320A of the first electrode 300A are respectively inserted into the pair of through holes. In order to hold the first electrode 300A on the first electrode fitting pin 133 in a state where the first electrode 300A is attached to the first electrode fitting pin 133, the O-ring 136 has a first electrode fitting. It is attached around the first electrode 300A on the side of the coupling pin 133. A cutout portion 143a is provided at the upper end of the second electrode fitting pin 143, and the curved portion of the second electrode 400A is accommodated or fitted in the cutout portion 143a. A pair of through holes are provided in the flange portion 130 around the lower end of the second electrode fitting pin 134. The upper end 410A and the lower end 420A of the second electrode 400A are respectively inserted into the pair of through holes. In order to hold the second electrode 400A on the second electrode fitting pin 134 in a state where the second electrode 400A is attached to the second electrode fitting pin 134, the O-ring 136 has a second electrode fitting. It is attached around the second electrode 400A on the side of the coupling pin 134.

A method for assembling the electrophoresis chip 1A according to the fourth embodiment will be described with reference to FIGS. As shown in FIG. 38 (A), in the first assembly step, the lower cap 130 is attached to the lower end opening of the gel containing tube 112 while the gel is contained, and the upper cap (gel) is attached to the upper end opening of the gel containing tube 112. A sealing plug 121 is attached, and the gel container 112 is sealed as shown in FIG. The upper cap 121 is rod-shaped and includes an annular knob 123 at the upper end thereof.

As shown in FIG. 39C, the knob 123 and the upper cap 121 are moved from the lower end opening of the buffer storage tube 110 into the buffer storage tube 110 with respect to the gel storage tube 112 in the state of FIG. insert. The gel storage tube 112 is moved in the direction of the buffer storage tube 110, and the upper end of the gel storage tube 112 is press-fitted into the lower end opening of the buffer storage tube 110 and integrated as shown in FIG. 39D, the entire knob 123 of the upper cap 121 is exposed from the upper end opening of the buffer housing tube 110.

Further, as shown in FIG. 40E, after the curved upper end 310A of the first electrode 300A is attached to the first electrode fitting pin 133, the first electrode 300A is bent by the O-ring 136. The upper end 310 </ b> A is fixed to the first electrode fitting pin 133. Similarly, after the curved upper end 410A of the second electrode 400A is attached to the second electrode fitting pin 134, the curved upper end 410A of the second electrode 400A is attached to the second electrode fitting by the O-ring 136. As shown in FIG. 40F, the assembly of the electrophoresis chip 1A of the fourth embodiment is completed. As shown in FIG. 40G, the electrophoresis chip 1A can include a support 160 that supports the lower portion of the first electrode 300A with respect to the lower portion of the gel housing tube 112.

The electrophoresis tool (electrophoresis chip), electrophoresis apparatus, and electrophoresis system of the first to fourth embodiments are not limited to electrophoresis of nucleic acids such as DNA, but are organic substances containing biologically related substances such as proteins. It can also be applied to electrophoresis. The vertical and horizontal movements of the electrophoresis chips 1, 1 ′, and 1 A of the electrophoresis chips 1, 1 ′, and 1 A according to the first to fourth embodiments can be automatically executed by the control unit according to a predetermined procedure. . Although the detection apparatus 700 of each embodiment detects fluorescence, it is not limited to this, The apparatus which detects chemiluminescence and can also detect light absorbency can also be used. In the case of detecting chemiluminescence, the excitation light source 705 and the excitation light optical fiber 710 can be omitted. In the case of detecting the absorbance, an absorbance photometer can be used in place of the photodetection element 740 such as PMT, and a colored labeled DNA can be used. It is also possible to provide an extraction / amplification apparatus that combines the electrophoresis apparatus or electrophoresis system of each embodiment with a DNA extraction mechanism using magnetic particles and / or an amplification mechanism that amplifies fragmented DNA by PCR or the like. . The electrophoresis apparatus, the extraction mechanism, and the amplification mechanism can automatically perform processing according to a predetermined procedure. The electrophoresis chips 1, 1 'and 1A according to the first to fourth embodiments can be automatically attached to and detached from the longitudinal movement unit 600 by an attaching / detaching mechanism. The light detection element 740 of each embodiment is not limited to the PMT, and an image pickup element such as a CCD or a CMOS can also be used.

In the electrophoresis system 3 of each embodiment, the error correction of the band position of each of the electrophoresis chips 1 to 1G arranged in parallel can be controlled to equalize the position of the electrophoresis band. The control unit of each embodiment observes the fluorescence-labeled DNA or the like mixed in the solution of the electrophoresis chip with the detection device 700 (fluorescence scanner) at all times, and arranges each electrophoresis so that it is aligned in a line at a certain substantially horizontal position. By individually turning on and off the chip voltage, the position can be controlled and DNA or the like can be accurately sorted or analyzed.

In the first embodiment, the second embodiment, and the fourth embodiment, the minus electrode 400 is provided on the upper side of the electrophoresis tube 100 and the plus electrode 300 is provided on the lower side to apply a potential to the gel. In this embodiment, the negative electrode 400 is provided on the lower side of the electrophoresis tube 100 and the positive electrode 300 is provided on the upper side to apply a potential to the gel. However, the present invention is not limited to this, and the negative electrode and the positive electrode are alternately arranged. Switching pulsed field gel electrophoresis (PFGE) can be used. As illustrated in FIGS. 12 and 14, the electrophoresis apparatus 2 includes a power supply unit 630 that is electrically connected to the power supply units 621 and 623. The power supply unit 630 can periodically change the electric field applied between the electrodes 300 and 400. More specifically, the power supply unit 630 can periodically reverse the direction in which the electric field between the electrodes 300 and 400 is applied. Furthermore, the power source unit 630 can periodically change the intensity of the electric field while periodically reversing the direction of the electric field between the electrodes 300 and 400. By performing PFGE, DNA having a large molecular weight (several tens of kbp to several Mbp) can be efficiently separated.

(Genetic marker analysis)
The electrophoresis system 3 of the first embodiment can also be used for gene marker analysis. As a genetic marker, single nucleotide polymorphism (SNP), restriction fragment length polymorphism (RFLP), or microsatellite can be used. Microsatellite exists in the genome. The electrophoresis system 3 of the first embodiment was used for gene marker analysis such as microsatellite analysis, and the like, and composed of repeating unit sequences of several bases (for example, 2 to 4 bases). The case will be described below.

As shown in FIG. 16, the electrophoresis system 3 of the first embodiment includes a plurality of electrophoresis chips 1 to 1B, and each of the electrophoresis chips 1 to 1B is used to perform electrophoresis of a plurality of different samples. Do. At the time of electrophoresis, the control unit performs measurement with the detection end 730 of the detection device 700 so that the positions of the reference bands (for example, molecular weight marker bands) of the samples are aligned, and the control unit controls the first electrode and the second electrode. Electrophoresis is performed by adjusting the voltage between the electrodes. As a result, a ladder (electrophoretic pattern) in which the positions of the reference bands are aligned can be obtained for different samples respectively loaded into the electrophoresis chips 1 to 1B.

Ladder information (arrangement or shape) can be obtained by scanning the detection end 730 while moving along the longitudinal direction of the electrophoresis tube. By analyzing the genetic markers contained in this ladder, it is possible to execute human individual identification, parent-child analysis, relative analysis, precision medicine (precision medicine), and the like. Moreover, since the electrophoresis system 3 of the first embodiment can align the positions of the reference bands, the analysis (comparison) of the ladder of each sample becomes easy. Furthermore, since the samples to be compared are electrophoresed in independent electrophoresis chips 1 to 1B using independent dispensers and wells, the possibility of contamination between different samples can be reduced. it can.

An embodiment of the electrophoresis system of the present invention will be described. In this example, the same parts as those in the first, second, and third embodiments are denoted by the same reference numerals, and description thereof will be omitted as appropriate. The present example has a configuration corresponding to the third embodiment. As shown in FIG. 32, the electrophoresis system 3 of this embodiment includes a sample container (well) 580 for storing a sample, a loading container 581 for storing a loading buffer, an electrophoresis container 582 for storing an electrophoresis buffer, and a cleaning liquid. And a cleaning container 583 for storing the container. In these containers, samples, reagents, and various buffers necessary for electrophoresis are dispensed in advance using a dispenser or the like. One or a plurality of containers 580, 581, 582, 583 can be provided. Containers 580, 581, 582, 583 are preferably placed on stage 800 (FIGS. 23-26). The sample is preferably DNA obtained from PCR. The loading buffer is a mixture of an electrophoresis buffer, a molecular weight marker, and a labeling die.

The loading die is a fluorescent dye for detecting double-stranded DNA, and for example, SYBER GREEN I can be used. The loading die can also contain Bromophenol Blue (BPB), Xylene Cyanole FF (XC), etc. so that the migration state can be visually confirmed. A molecular weight marker is a DNA fragment of a known size for estimating the size of DNA to be analyzed. The molecular weight marker is electrophoresed simultaneously with a sample such as DNA. The molecular weight marker preferably comprises two sizes, and the DNA size to be analyzed must be between the sizes of the two molecular weight markers. As the electrophoresis buffer, the type and concentration of the electrophoresis buffer that is optimal for the gel composition and the size of the DNA to be separated can be selected. As the electrophoresis buffer, for example, a TAE buffer or a TBE buffer can be used. The electrophoresis buffer can also be used as a cleaning solution for the tip of the electrophoresis tube 100 (capillary column).

The steps 1) to 3) executed by the electrophoresis method of this embodiment will be described with reference to FIG. First, in step 1), sample adjustment is performed. Specifically, in a state where the sample (for example, 5 μl) in the sample container 580 is sucked into the dispensing tip 611 attached to the dispensing device 610, the dispensing device 610 is moved and discharged to the loading container 581. Using the dispenser 610, the liquid in the loading container 581 is repeatedly sucked and discharged, and the sample in the loading container 581 and the loading buffer are stirred and mixed thoroughly.

In step 2), the sample is loaded using the electrophoresis chip 1 (1 '). Specifically, the lower ends of the electrophoresis tube 100 and the negative electrode (first electrode) 300 are immersed in a mixed solution of the sample and the loading buffer in the loading container 581. In this state, a predetermined voltage is applied for a predetermined time, and the sample is infiltrated (loaded) into the lower end of the gel. The predetermined time can be preferably 1 to 5 seconds, more preferably 2 to 3 seconds. The predetermined voltage is preferably 50 to 150V, more preferably 80 to 120V, and still more preferably about 100V. The amount of sample to be electrophoresed is determined by the predetermined time applied at this time. Increasing the loading amount of the sample can increase the fluorescence intensity, but the resolution of the DNA size of the sample decreases.

In step 3), electrophoresis and analysis of the sample are performed using the electrophoresis chip 1 (1 '). Specifically, the electrophoresis chip 1 (1 ′) loaded with the sample in the loading container 581 is moved to the cleaning container 583 according to the arrow A 1. The lower end of the electrophoresis tube 100 and the negative electrode 300 can be washed into and removed from the cleaning liquid in the cleaning container 583 by washing. By washing the lower end portion, DNA that has not penetrated into the gel can be washed away and the influence thereof can be eliminated. After this washing, the electrophoresis chip 1 (1 ') is moved to the electrophoresis container 582 according to the arrow A2. The lower ends of the electrophoresis tube 100 and the negative electrode 300 of the electrophoresis chip 1 (1 ′) are immersed in the electrophoresis buffer in the electrophoresis container 582. Then, the predetermined voltage is applied between the minus electrode 300 and the plus electrode 400 of the electrophoresis tube 100, and electrophoresis is performed in the electrophoresis tube 100. Since samples such as DNA are negatively charged, they move upward from the bottom of the electrophoresis tube 100 by electrophoresis. Note that when the applied voltage is increased, the electrophoresis time can be shortened, but the electrophoresis pattern is likely to be disturbed due to heat generation or the like.

An electrophoretic ladder is obtained by step 3). The analysis of the electrophoresis ladder will be described with reference to FIG. FIG. 35 shows a state in which the sample S or the like is electrophoresed in the electrophoresis tube 100. In the sample S, two kinds of markers having a known size (molecular weight) are mixed in advance at the time of sample preparation. The two types of markers, and a low molecular weight marker M _L, higher than the molecular weight of the sample DNA to be detected (large) and a high molecular weight marker M _H having a molecular weight selected with a lower molecular weight (small) molecular weight of the sample DNA to be detected Is done. As shown in FIG. 35, a detection end 730 of a detection device (fluorescence scanner) 700 is disposed on the side surface of the electrophoresis tube 100.

Then, two types of markers M _L and M _H are electrophoresed simultaneously with the sample S. As shown in FIG. 35, when the time T from the electrophoresis start has elapsed, the movement distance D _L of the low molecular weight marker M _L, the travel distance D _S of the sample S, the movement distance D _H of the high molecular weight marker M _H, a measurement To do. As a measurement method of the movement distance, by measuring the fluorescence of each marker and sample while moving the detection end 730 shown on the right side of FIG. 35 from the lower side to the upper side in the longitudinal direction of the electrophoresis tube 100, Each moving distance D can be measured. Moving distance D _L of the low molecular weight marker M _L, the molecular weight of the moving distance D _H, low molecular weight marker M _L high molecular weight marker M _H, molecular weight of the high molecular weight marker M _H, with a travel time T, DNA size and electrophoresis A relational expression of speed can be obtained. By applying the position of the sample S between the two markers to the previously obtained relational expression, the size of the sample DNA can be estimated.

The size of the analysis to DNA contained in the sample (molecular weight), 2 types of markers M _L, must be located between the M _H. The two types of markers M _L, the larger the difference in size of the M _H (molecular weight), because the estimation accuracy is lowered, preferably, with respect to the molecular weight of the low molecular weight marker M _L, the high molecular weight marker M _H The molecular weight can be set to about 2 to 3 times.
The small molecule marker should be as large as easily distinguishable from the primer-dimer contained in sample S.

The size of the sample DNA can also be estimated by measuring the movement time without measuring the movement distance. The position of the detection end 730 shown on the left side of FIG. 35 is fixed to a known detection position P _D. Low molecular weight markers M _L, sample S, molecular weight marker M _H is, by measuring the passing time through the detection position P _D respectively, each passing time, the molecular weight of the low molecular weight marker M _L, high molecular weight marker M _H The size of the sample DNA can be estimated from the relational expression of the molecular weight. In addition, although the present Example was demonstrated as electrophoresis which moves the electrophoresis tube 100 from the bottom to the top based on 3rd Embodiment, it is not limited to this, Like 1st and 2nd Embodiment, It is also possible to apply to electrophoresis in which the electrophoresis tube 100 moves from top to bottom.

1,1 ′ electrophoresis chip 2 electrophoresis apparatus 3 electrophoresis system 100 electrophoresis tube 110 buffer housing tube 112 gel housing tube 200 holder 210 plus electrode housing portion 220 minus electrode housing portion 300 plus electrode 400 minus electrode 500 cartridge 600 longitudinal movement Unit 700 Detection device 730 Detection end 800 Stage 900 Horizontal movement unit 1200 Holder 1210 Positive electrode accommodating portion 1220 Negative electrode accommodating portion

Claims (51)

1. An electrophoresis tube containing an electrophoresis gel and a buffer, a first electrode for applying a positive or negative potential to the lower side of the electrophoresis tube, and the other positive or negative side on the upper side of the electrophoresis tube An electrophoretic tool comprising: a second electrode for applying a potential of
   An electrophoresis device comprising a holder that integrally holds the electrophoresis tube and the first electrode.
2. 2. The electrophoresis tool according to claim 1, wherein the holder integrally holds the electrophoresis tube, the first electrode, and the second electrode.
3. The electrophoretic device according to claim 1 or 2,
   The holder is an electrophoresis tool that holds an upper part of the electrophoresis tube.
4. The electrophoresis device according to any one of claims 1 to 3,
   The holder includes an electrophoretic device including a first electrode housing portion that houses the first electrode and a second electrode housing portion that houses the second electrode.
5. The electrophoretic device according to claim 4,
   An electrophoretic device comprising a hinge portion connecting the first electrode housing portion and the second electrode housing portion.
6. The electrophoretic device according to claim 4,
   The electrophoretic tool, wherein the first electrode housing portion and the second electrode housing portion are separable.
7. The electrophoretic device according to claim 6,
   The first electrode housing part has an opening into which the second electrode housing part can be inserted.
8. The electrophoretic device according to any one of claims 1 to 7,
   The second electrode extends along the electrophoresis tube from the upper side to the lower side of the electrophoresis tube.
9. The electrophoretic device according to claim 8,
   The holder includes an extension that extends to the lower side of the electrophoresis tube along the second electrode.
10. The electrophoresis tool according to claim 9,
    The extension part is an electrophoresis tool including a holding part that holds the electrophoresis tube.
11. The electrophoretic device according to any one of claims 1 to 10,
    The holder includes an accommodation groove that accommodates the second electrode.
12. The electrophoretic device according to any one of claims 1 to 11,
    The electrophoresis tool, wherein a lower end of the first electrode is inserted into the electrophoresis tube.
13. The electrophoresis device according to claim 1,
    The electrophoretic device, wherein the holder includes a first fitting body integrally formed with the electrophoresis tube so as to be fitted with the first electrode.
14. The electrophoretic device according to claim 15,
    The holder includes a second fitting body integrally formed with the electrophoresis tube so as to fit with the second electrode.
15. The electrophoresis device according to claim 13 or 14,
    The holder is an electrophoresis tool provided on an upper part of the electrophoresis tube.
16. The electrophoresis intelligent tool according to any one of claims 1 to 14,
    The electrophoresis tube is an electrophoresis tool including a buffer storage tube that stores a buffer and a gel storage tube that stores the gel.
17. The electrophoretic device according to claim 16,
    The electrophoresis tool, wherein a lower end of the first electrode is disposed in the buffer housing tube.
18. An electrophoresis apparatus comprising:
    A control unit for controlling electrophoresis;
    An electrophoretic device according to any one of claims 1 to 17,
    A first power feeding unit that feeds power to the first electrode of the electrophoresis tool;
    A second power feeding section for feeding power to the second electrode of the electrophoresis tool;
    A dispenser;
    A well into which the second electrode of the electrophoresis tool and the lower end of the electrophoresis tube are inserted;
    An electrophoresis apparatus comprising: the electrophoresis device and / or a moving unit capable of moving the dispenser three-dimensionally.
19. The electrophoresis apparatus according to claim 18, wherein
    An electrophoretic apparatus comprising a power supply unit that periodically changes an electric field applied between the first electrode and the second electrode in order to perform pulse field gel electrophoresis.
20. The electrophoresis apparatus according to claim 19,
    The power supply unit is an electrophoresis apparatus that periodically inverts the direction of the electric field.
21. The electrophoresis apparatus according to claim 18 or 19,
    The electrophoretic device, wherein the power supply unit periodically changes the intensity of the electric field.
22. The electrophoresis apparatus according to any one of claims 18 to 21,
    The moving unit includes a vertical moving unit that moves the electrophoretic instrument and / or the dispenser substantially linearly in a vertical direction, and a linear movement unit that moves the electrophoretic instrument and / or the dispenser substantially linearly in a horizontal direction. An electrophoretic device comprising a laterally moving unit that moves.
23. The electrophoresis apparatus according to claim 22,
    The lateral movement unit includes the vertical movement unit, and moves the vertical movement unit in a horizontal direction.
24. The electrophoresis apparatus according to any one of claims 18 to 23,
    The well is a plurality of wells;
    An electrophoresis apparatus, wherein the moving unit moves the electrophoretic tool to separate the plurality of bands into the plurality of wells.
25. The electrophoresis apparatus according to claim 24, wherein
    When the moving unit moves the electrophoresis tool, the control unit moves the electrophoresis tool above the well by the moving unit.
26. The electrophoresis apparatus according to claim 24 or 25,
    When the moving unit moves the electrophoresis tool,
    The controller is an electrophoretic device that stops power supply from the first power supply unit and the second power supply unit to the first electrode and the second electrode.
27. The electrophoresis apparatus according to any one of claims 18 to 26,
    The well includes an electrophoresis apparatus including a plurality of containers arranged in an annular shape.
28. The electrophoresis apparatus according to claim 27,
    The well includes an electrophoresis apparatus including a buffer container surrounded by the plurality of containers.
29. The electrophoresis apparatus according to claim 27 or 28,
    An electrophoresis apparatus comprising a well rotation mechanism for rotating the well.
30. The electrophoresis apparatus according to claim 29,
    The electrophoresis apparatus, wherein the well rotation mechanism switches the plurality of sorting units by rotating the well.
31. The electrophoresis apparatus according to claim 30, wherein
    When the well rotation mechanism rotates the well, the electrophoresis tool moves upward away from the well by the moving unit.
32. The electrophoresis apparatus according to any one of claims 29 to 31,
    When the well rotation mechanism rotates the well,
    The controller is an electrophoretic device that stops power supply from the first power supply unit and the second power supply unit to the first electrode and the second electrode.
33. The electrophoresis apparatus according to claim 22 or 23,
    The dispenser is an electrophoresis apparatus connected to the longitudinal movement unit.
34. The electrophoresis apparatus according to any one of claims 22, 23, and 33,
    The longitudinal movement unit is an electrophoretic device that integrally moves the first power feeding unit and the second power feeding unit.
35. The electrophoresis apparatus according to any one of claims 18 to 34,
    An electrophoresis apparatus comprising a detection device for detecting a band or a ladder generated when a sample flows through the gel of the electrophoresis tube.
36. 36. The electrophoresis apparatus according to claim 35,
    The detection apparatus includes an detection end facing the electrophoresis tube of the electrophoresis tool, and a detection end moving unit that moves the detection end in a three-dimensional manner.
37. The electrophoresis apparatus according to claim 36,
    The detection end moving unit moves the detection end along the longitudinal direction of the electrophoresis tube.
38. The electrophoresis apparatus according to claim 36 or 37,
    The detection end moving unit moves the detection end in a state of facing the side surface of the electrophoresis tube or the well.
39. The electrophoresis apparatus according to any one of claims 36 to 38,
    The detection end moving unit moves the detection end in a direction approaching or moving away from a side surface of the electrophoresis tube or the well with the detection end facing the electrophoresis tube or the well. An electrophoresis device.
40. The electrophoresis apparatus according to any one of claims 36 to 37,
    The detection end is an electrophoresis apparatus that holds an irradiation end of an optical fiber for excitation light and a light reception end of an optical fiber for fluorescence reception.
41. The electrophoresis apparatus according to any one of claims 36 to 40,
    The electrophoretic device, wherein the detection end portion includes a curved surface along a contour of a side surface of the well.
42. The electrophoresis apparatus according to any one of claims 36 to 41,
    An electrophoresis apparatus, wherein the detection apparatus detects positions of a plurality of bands separated from a sample being electrophoresed.
43. The electrophoresis apparatus according to any one of claims 18 to 42,
    An electrophoresis apparatus comprising a nucleic acid extraction mechanism using magnetic particles.
44. The electrophoresis apparatus according to any one of claims 18 to 43,
    An electrophoresis apparatus comprising an amplification mechanism for amplifying a nucleic acid.
45. An electrophoresis system comprising a plurality of the electrophoresis apparatuses according to any one of claims 18 to 44.
46. The electrophoresis system according to claim 45,
    The control unit detects a position of a band separated from a sample using the detection device for each of the plurality of electrophoresis tools, and based on the detection position of the band, An electrophoresis system that controls movement of the band in each electrophoresis tool by controlling power feeding to each of the second electrode and the first electrode of each electrophoresis tool.
47. The electrophoresis system according to claim 46,
    The electrophoretic system, wherein the control unit controls power supply to the first electrode and the second electrode so as to align the positions of the bands in each of the plurality of electrophoretic tools.
48. An electrophoresis method for performing electrophoresis of a sample using the electrophoresis apparatus according to any one of claims 18 to 44.
49. An electrophoresis method for performing parallel processing of electrophoresis of a plurality of samples using the electrophoresis system according to any one of claims 44 to 47.
50. The electrophoresis method according to claim 48 or 49,
    The method wherein the sample is a biological material.
51. The electrophoresis method according to any one of claims 48 to 50,
    The method, wherein the biological substance is DNA, RNA, or protein.

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