JP2000009690A - Solution filling apparatus for capillary electrophoresis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means by which both an operation to fill a polymer solution into a plurality of capillaries and an operation to immerse tip parts of the capillaries into another solution are performed automatically. SOLUTION: Tip parts 52 of a plurality of capillaries 51 are passed through a plurality of conical connecting jigs 53 fixed to a holding implement 54. An airtight pressure-resistant block in which a plurality of conical holes complementary to the connecting jigs 53 are opened is filled with a polymer solution. The tip parts 52 are inserted into the holes. The connecting jigs 53 are made to collide with the holes in the pressure-resistant block. The connecting jigs 53 are deformed. The holes are sealed airtightly. The pressure at the inside of the pressure-resistant block is increased by a pump. The polymer solution is filled into the capillaries 51. The capillaries 51 are moved to a solution tank, and their tip parts 52 are immersed in an arbitrary solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an apparatus for separating and analyzing DNA, RNA or protein. In particular, the present invention relates to an effective method for determining the nucleotide sequence of DNA or RNA, or measuring the polymorphism of a restriction enzyme fragment based on the diversity of individual nucleotide sequences.

[0002]

2. Description of the Related Art Analysis techniques for DNA, RNA and the like are becoming increasingly important in the fields of medicine and biology, including genetic analysis and genetic diagnosis. Particularly in recent years, high-speed, high-throughput DNA analyzers have been developed in connection with the genome analysis plan.

[0003] DNA analysis is carried out by separating the molecular weight of a fluorescently labeled DNA sample by gel electrophoresis and detecting the laser-induced fluorescence. For gel electrophoresis, a flat gel obtained by polymerizing acrylamide between two glass plates with a space of about 0.3 mm is often used. The DNA sample injected into the upper end of the slab gel is applied to the upper and lower ends of the slab gel by applying an electric field with the upper end being the cathode and the lower end being the anode, so that the negatively charged DNA sample is migrated toward the lower end while being separated by molecular weight. . All the migration paths are illuminated at once from the side of the flat gel with the laser at the position where the electrophoresis has been carried out at a fixed distance, and the separation component of the fluorescently labeled sample passing through the laser irradiation part is excited. Fluorescence exposure is performed at fixed time intervals, and continuous cycle measurement is performed. By analyzing this result, the DNA base sequence is determined.

Recently, instead of a flat gel, a polymerized polymer solution is filled in a fused quartz capillary tube having an inner diameter of about 75 μm,
Capillary electrophoresis using a polymer solution as a separation medium has been commercialized and commercialized from Perkin Elmer (Perkin Elm)
er ABI Prism 310 Genetic Analyzer) (hereinafter referred to as ABI 310). D injected from one end of the capillary
For NA samples, DNA with negative charge is applied to both ends of the capillary by applying an electric field with the sample injection end as the cathode and the other end as the anode.
The sample is electrophoresed in the capillary from the sample injection end to the sample elution end (the other end) while being subjected to molecular weight separation, and the same analysis as in flat plate gel electrophoresis is performed. Capillary electrophoresis
Compared to slab gel electrophoresis, a high electric field can be applied due to the high electric resistance and high heat dissipation effect, so this method is capable of high speed and high separation (Analytical Che
mistry 62, 900 (1990)).

The ABI 310 uses one capillary tube, irradiates the inside of the capillary near the elution end with a laser, and performs on-column measurement for fluorescence detection. Since the entire outer surface of the capillary is coated with polyimide, the coating at the detection position is removed to form a window with exposed glass (US Pat. No. 5,312,535 (May 17, 1994)). This position is irradiated with a laser, and the separated components of the fluorescently labeled sample that migrates inside the capillary by electrophoresis are excited when passing through the beam, and the emission fluorescence is measured and analyzed in a continuous cycle to determine the DNA base sequence. ing.

[0006] In capillary electrophoresis using a polymer solution as a separation medium, the polymer solution inside the capillary must be exchanged for each electrophoresis in order to avoid deterioration of the separation medium and mixing of different samples. In the ABI 310, the polymer solution inside the capillary can be refilled every time the electrophoretic analysis of one sample is completed, so that a large number of samples can be sequentially and automatically analyzed using the same capillary repeatedly. The method of filling the ABI 310 with a polymer solution and the method of measuring electrophoresis will be described below.

[0007] The polymer solution is a viscous fluid having a viscosity of more than 100 times that of water, and the capillary is a capillary having an inner diameter of 75 µm or less. Therefore, the capillary immersed in the polymer solution is used to fill the capillary with the polymer solution. A high pressure of 10 atm or more is applied to one end of the. The sample elution end of the capillary is pressure-resistant connected to a gas tight syringe filled with a polymer solution via an acrylic pressure block filled with the polymer solution. The pressure-resistant block can also be connected to the anode buffer tank via a pressure-resistant valve.

With the pressure-resistant valve closed, the pressure inside the pressure-resistant block is increased by mechanically pushing the piston of the gas-tight syringe, and the polymer solution is eluted from the sample (on the anode side).
From the sample injection end (cathode side). After completion of filling, the capillary sample elution end is electrically connected to the anode buffer solution tank by opening the pressure-resistant valve, and the capillary sample injection end is immersed in the cathode buffer solution tank. Capillary electrophoresis is performed by applying an electric field between both buffer tanks. The sample injection is performed by immersing the capillary sample injection end in the sample solution and applying an electric field between the anode buffer solution tank and the sample solution for a certain period of time. After the electrophoresis measurement, the pressure-resistant valve of the pressure-resistant block is closed again, and the same cycle of polymer filling, sample injection, and electrophoresis measurement as described above is repeated.

During the execution of this cycle, the capillary sample elution end is connected to the pressure-resistant block from the beginning to the end. The pressure-resistant connection is performed via a connection jig called a plastic ferrule. The ferrule is a male screw with a pointed cone, with a small hole in the center just enough to allow the capillary to penetrate. The connection part of the pressure-resistant block has a female screw hole which is tapered so as to be compatible with the ferrule, and the end thereof communicates with the polymer inside the pressure-resistant block.

When this screw is tightened with the capillary penetrating the ferrule, the tip of the ferrule collides with the taper of the pressure-resistant block, and the tip of the ferrule is deformed. Thus, the gap between the ferrule and the capillary and the gap between the ferrule and the pressure-resistant block are sealed. Because this seal is fixed by the screw mechanism,
Even if the inside of the pressure-resistant block is in a high pressure state of several tens of atmospheres, the polymer solution does not leak and a pressure-resistant connection is formed. As described above, the ABI 310 repeatedly uses the same capillary without exchanging it, and sequentially samples a large number of samples.
Automatic measurement allows DN with low cost and low labor
A Can analyze.

However, the ABI 310 has a low throughput because only one capillary can be measured at a time. In order to make this apparatus a high-throughput automatic analyzer, it must be developed into a capillary array apparatus that can simultaneously measure many capillaries. The capillary array device mainly has the following two development examples.

One development example is a capillary array scan method (Nature, 359, 167 (1992)).
A plurality of capillaries are irradiated with laser one by one in order, and on-column fluorescence measurement is performed. This device has a confocal structure in which the position where the laser beam is most converged in the capillary and the position of the light source incident on the fluorescence measurement device are taken, and each capillary can be measured independently. The laser irradiation and the fluorescence receiving optical system are fixed, and the stage holding the capillary array reciprocates in a one-dimensional direction, thereby sequentially irradiating each capillary with laser.

In the present method, since the fluorescence measurement is performed one by one in order of the capillaries, the fluorescence exposure time for one capillary is reduced as compared with the ordinary on-column measurement. When n capillary arrays are used, the maximum fluorescence exposure time per line is 1 / n, and the glass part of the capillary through which the sample separation component does not actually pass is also scanned. I will. As a result, there arises a problem that the fluorescence detection sensitivity is reduced. In addition, the time interval between adjacent peaks in the separated component electrophoresis pattern of the sample becomes smaller as the analysis speed increases, but if the time required for one scan becomes too large to be ignored, the separated component electrophoresis pattern A problem arises in that the separation power of the polymer decreases. Furthermore, this apparatus has a stage movable section for scanning,
It has a structure where failures occur frequently.

Another development example is a capillary array sheath flow method (Nature, 361, 565-566 (1993), Japanese Patent Laid-Open No. 6-138037). In this device, the sample injection end of the capillary array arranged on a plane is immersed in the cathode buffer solution tank,
The sample elution end shall be immersed in the anode buffer tank and the cell which is the fluorescence measurement part, and the sample components separated by electrophoresis will be eluted into the buffer as it is, and the buffer part without this capillary will be irradiated with laser. The fluorescence detection of the sample component eluted in is performed. At this time, by slowly flowing the buffer solution in the cell in the electrophoresis direction, the separated components eluted from different capillary gels are mixed with each other in the buffer solution, or two components separated in one capillary gel are separated. The components are not mixed in the buffer.

The flow of the sample component eluted from the capillary in a sheath-like manner with a buffer solution is called a sheath flow, and the cell in which the buffer solution flows is called a sheath flow cell. Since the laser irradiates the buffer portion where there is no capillary, the problem of laser beam scattering on the capillary surface is avoided, and the components eluted from multiple capillaries can be excited collectively, enabling simultaneous fluorescence analysis. I have. Therefore, this method has higher fluorescence detection sensitivity and can measure more capillaries simultaneously than the capillary array scanning method.

That is, if the polymer filling device and the capillary array sheath flow method can be combined, a DNA analyzer with the lowest cost, high sensitivity, and high throughput can be obtained.

[0017]

Generally, in capillary electrophoresis using a polymer solution, it is necessary to wash the inner surface of the capillary with a reagent other than the polymer solution between one electrophoresis analysis and the next electrophoresis analysis. (Analytical Chemistry 67, 1913)
(1995)). If this is neglected, the state of charge on the inner surface of the capillary changes with time, and this may increase the electroosmotic flow and reduce the electrophoretic separation performance.

However, in the ABI 310, the elution end of the capillary sample is pressure-resistant connected to the pressure-resistant block from beginning to end, and the inside of the capillary is always filled with the polymer solution. . Also,
In the ABI 310, the elution end of the capillary sample is always pressure-resistant connected to the pressure-resistant block, so that it is not possible to perform electrophoresis by immersing the elution end of the sample in an arbitrary buffer. In either case, it is necessary to attach and detach the pressure-resistant connection, but the pressure-resistant connection means to the pressure-resistant block of the capillary used in ABI 310 is:
Since it is a screw-fixed ferrule, it is difficult for the machine to automatically attach and detach it.

On the other hand, it is impossible to apply the polymer filling device of ABI 310 directly to the capillary array sheath flow system for the following reasons. In the ABI 310, the sample elution end of the capillary is immersed in the polymer solution, and this part is set to a high pressure of 10 atm or more, so that the polymer solution is filled in the direction from the sample elution end to the sample injection end of the capillary. In the sheath flow method, the sample elution end of the capillary is fixed in a sheath flow cell filled with a buffer solution.

Therefore, in order to adopt the polymer filling method of ABI 310, after replacing the buffer solution in the sheath flow cell with the polymer solution to be filled, a high pressure of 10 atm or more must be applied to the inside of the sheath flow cell. No. Since existing sheath flow cells do not have such pressure resistance, this polymer filling method is not feasible. Even if there is a sheath flow cell having pressure resistance, it is necessary to replace the buffer inside the sheath flow cell with the polymer solution every time the polymer solution is filled, and to return the buffer to the buffer after filling. This is an operation that requires a lot of time, and is contrary to the high speed characteristic of capillary electrophoresis. In addition, a large amount of the polymer solution and the buffer solution are wasted, which increases the cost.

It is possible in principle to fill the polymer solution from the reverse direction of the capillary array using the polymer filling device of ABI 310. That is, it is possible to fill the polymer from the sample injection end of the capillary array to the sample elution end arranged in the sheath flow cell, since the sheath flow cell does not require pressure resistance. A female screw hole is made in the pressure-resistant block in a straight line at an interval equal to the arrangement interval of the sample injection ends of the capillary array arranged on a plane, and the sample injection end of each capillary is connected to each of the pressure-resistant blocks via a screw-type ferrule. Connect with pressure to the female screw hole. At this time, the number of ferrules to be connected with pressure is required to be the same as the number of capillaries in the capillary array. The pressure inside the pressure-resistant block is increased by mechanically pressing the piston of the gas tight syringe connected to each capillary via the pressure-resistant block, and the polymer solution is filled from the sample injection end of each capillary to the sample elution end. it can. However, this method has the following two problems.

In order to perform a capillary electrophoresis analysis,
After filling the polymer solution, the capillary sample injection end is immersed in the sample solution to perform sample injection, and then the capillary sample injection end is immersed in the cathode buffer bath to perform electrophoresis. That is, it is necessary to attach / detach the sample injection end of each capillary to / from the pressure-resistant block. Since the means for connecting the pressure-resistant block of the capillary used in the ABI 310 to the pressure-resistant block is a screw-fixed ferrule, it is difficult for the machine to automatically attach and detach it. Considering simultaneous attachment and detachment of screw-type ferrules corresponding to the number of capillaries in the capillary array, its automation is more difficult.

Another problem is that, since the pressure-resistant connecting means is a screw-fixed ferrule, the arrangement interval of the female screw holes formed in the pressure-resistant block in a straight line becomes large.
Since the arrangement interval between the sample injection ends of the capillary array arranged on a plane is equal to this, the arrangement interval is also large. on the other hand,
The arrangement interval of the sample elution ends of the capillary array arranged on a plane is as small as possible in order to perform high-sensitivity fluorescence detection inside the sheath flow cell.

That is, the arrangement interval of the capillary array increases from the sample elution end to the sample injection end. The greater the extent of this spread, the longer the overall length of each capillary in the capillary array will be from the centrally located capillary to the end located capillary.

Since the diameter of the ferrule used in the ABI 310 is about 10 mm, if a female screw hole is formed on the pressure-resistant block in a straight line at intervals of 10 mm, the total width of the sample injection end of the 48 capillary arrays is Up to 480 mm. In contrast, the spacing between the sample elution ends of the capillary array was 0.4
Since it is about mm, the total width is about 19 mm, which is sufficiently small. If the total length of the capillary arranged at the center of the capillary array is 240 mm, the total length of the capillaries arranged at both ends is about √2 times as large as 340 mm. In other words, even though they are in the same capillary array, they are analyzed under different electrophoresis conditions for each capillary,
Very inconvenient. It is also difficult to manufacture a huge pressure-resistant block as large as 480 mm.

An object of the present invention is to solve the above two problems, to enable the sample injection end of the capillary array to be automatically attached to and detached from the pressure-resistant block, and to reduce the arrangement interval between the sample injection ends. An object of the present invention is to provide a polymer solution filling apparatus for reducing the difference in the total length of each capillary in a capillary array.

[0027]

The filling device of the present invention does not use a screw mechanism between the connection jig (ferrule) and the pressure-resistant block for the pressure-resistant connection means of the capillary to the pressure-resistant block. The connection jig has a pointed conical tip, and has a small hole in the center just enough to allow the capillary to penetrate. The connection portion of the pressure-resistant block has a tapered hole that matches the conically pointed portion of the connection jig, and is connected to the polymer solution inside the pressure-resistant block.
The pressing jig is connected to the connection jig so that the connection jig can be mechanically pressed against the hole of the connection portion of the pressure-resistant block.

Since this connecting means does not need to be attached or detached,
It may be one using a screw mechanism or one using an adhesive. When a capillary is passed through the connection jig and a load is applied to the connection jig using a pressing jig and the connection jig is pressed against the hole of the connection part of the pressure-resistant block, the tip of the connection jig becomes the taper of the pressure-resistant block. The collision causes the tip of the connection jig to be deformed.
Thereby, the gap between the capillary and the connection jig and the gap between the connection jig and the pressure-resistant block are sealed, and a pressure-resistant connection is formed. The pressing jig may be, for example, a motor having a linear drive mechanism. By simply controlling the motor, a load can be applied to the connection jig, and conversely, the connection jig can be pulled back.

That is, it is also possible to automatically connect the capillary and the pressure-resistant block to each other with pressure or disconnect the connection so that the sample injection end of the capillary is immersed in another solution tank.
Also, if a large number of connection jigs are combined with one pressing jig, they can be controlled collectively, so even for a capillary array that handles a large number of capillaries, the same method can be applied to the pressure-resistant block. The attachment and detachment can be performed automatically.

Further, according to the present invention, since a screw mechanism is not required for the connection jig and the pressure-resistant block, the hole at the connection portion between the connection jig and the pressure-resistant block can be made very small as compared with the case of the ABI 310. Therefore, a large number of connection jigs linearly coupled to one pressing jig and a large number of connection portion holes formed linearly on the pressure-resistant block can be arranged at very small arrangement intervals. . In other words, each capillary can be pressure-resistant connected to a pressure-resistant block using these jigs while the arrangement interval between the sample injection ends of the capillary array arranged on a plane is extremely small. For example, if the diameter of the connection jig is 2 mm and the arrangement interval between the sample injection ends of the 48 capillary arrays is 3 mm, the overall width of the sample injection end is reduced to 144 mm. Assuming that the total length of the capillaries arranged at the center of the capillary array is 240 mm, the total length of the capillaries arranged at both ends is 251 mm, and the difference in capillary length in the same capillary array is less than 4%. Analysis can be performed under the same electrophoresis conditions.

[0031]

(Embodiment 1) A polymer solution filling apparatus according to the present invention is manufactured by using Perkin Elmer's product ABI 310.
The present invention is applied to a single capillary electrophoresis apparatus for on-column fluorescence detection similar to the above.

The electrophoresis buffer was 1 × TBE (89 mM (mmol / liter) containing 3.5 M (mol / liter) of urea).
droxymethyl) aminomethane), 89mM boric acid (boric aci
d), use 2 mM ethylenediaminetetraacetic acid (EDTA). The polymer solution of the electrophoresis separation medium is 6% T, 0% C linear polyacrylamide, 1 × TB containing 3.5 M urea.
Use the one dissolved in E. The viscosity of this polymer solution is μ = 4900 CP = 4.9 N · sec / m ² (Analytical Chem.
istry 67, 1913 (1995)).

Outer diameter R = 0.375 mm, inner diameter r = 0.0075 mm, overall length
1 from one end of L = 350 mm capillary (Polymicro)
The polyimide coating at the position of 00 mm is removed over a width of 10 mm, and is used as a fluorescence detection position. During electrophoresis, the capillary end farther from the fluorescence detection position is the sample injection end, and the capillary end closer to the fluorescence detection position is the sample elution end. Therefore, the effective length of capillary electrophoresis is l = 250 mm.

As shown in FIG. 1, the fluorescence detection position of the capillary 1 is set on the capillary holder 2 of the fluorescence detector, and the central portion of the capillary is fixed on the temperature control plate 3 in an inverted U shape. The lower right end of the capillary is the sample injection end 4, and the lower left end is the sample elution end 5. The capillaries, including both ends, are immobilized throughout the polymer filling, sample injection, and electrophoresis steps. The temperature control plate 3 can arbitrarily control the capillary temperature during electrophoresis from a position 50 mm from the sample injection end 4 of the capillary to a position from the fluorescence detection position to a position 50 mm from the sample injection end. Here, the temperature control plate 3 is set to 40 ° C. throughout the entire process.

In the fluorescence detection, a laser is applied to the fluorescence detection position of the capillary vertically from the rear of the paper of FIG. 1 to the paper, and the emitted fluorescent light is received by the fluorescence detector from the rear of the paper. The laser uses 488 nm and 514.5 nm argon ion lasers. In a fluorescence detector, fluorescence is wavelength-dispersed by a diffraction grating, and various types of fluorescence are detected at once by a CCD camera. A sample injection device 6 is installed at the sample injection end 4 of the capillary, and a polymer solution filling device 7 is installed at the sample elution end 5.

FIG. 2 shows a configuration diagram of the polymer solution filling device 7. The connecting jig 8 is made of PEEK (polyetheretherketone), has a conical shape with a bottom surface diameter of 10 mm and a height of 20 mm, and has a hole of 0.5 mm diameter at the center. In order to hold the connection jig 8 in a space 50 mm below the capillary holder 2, the connection jig 8 is connected to the holder 9 by an adhesive. The holder 9 is made of aluminum, and a hole having a diameter of 5 mm is formed at a position coaxial with the hole of the connection jig 8 so that the capillary can pass therethrough. The sample elution end 5 of the capillary 1 is passed through the holes of the holder 9 and the connection jig 8, and the end of the capillary 1 is connected to the connection jig 8.
20 mm from the tip of The pressure-resistant block 10
It is a cylinder made of acrylic, with a bottom diameter of 20 mm and a height of 40 mm. A 1 mm diameter hole penetrates the center, and a conical hole with the same shape as the connection jig is opened in the center of the top surface. A gas tight syringe 11 having a capacity of 1 ml is pressure-resistant connected to the center of the bottom surface.

The inside of the gas tight syringe 11 and the hole of 1 mm in diameter of the pressure-resistant block 10 are filled with the polymer solution 12 (6% T linear
Polyacrylamide). The pressure-resistant block 10 and the gas tight syringe 11 are movable in the left and right direction (X direction) and the vertical direction (Z direction) in FIG. 2 by connecting the piston 13 of the gas tight syringe 11 to the XZ stage 14.

The movable stage of the XZ stage 14 in the Z-axis direction has a built-in load sensor, and can measure the load applied in the Z-axis direction. The X axis of the XZ stage 14 is moved so that the center of the pressure-resistant block 10 is located immediately below the sample elution end 5 of the capillary 1. Subsequently, the pressure block 10 and the gas tight syringe 11 are moved closer to the capillary elution end and the connecting jig by moving the Z axis upward. The sample elution end 5 of the capillary 1 is guided to the tapered hole of the pressure-resistant block 10 and is filled with the polymer solution 12 as shown in FIG.
Is inserted into the hole. When the Z-axis continues to move upward, the connection jig 8 and the tapered hole of the pressure-resistant block 10 collide.

FIG. 3 shows a state of the collision at this time. FIG.
When the state before the collision shown in FIG. 3A is shifted to the state after the collision shown in FIG. 3B, the tip of the connecting jig 8 is deformed and crushed, and the diameter 1 of the pressure-resistant block 10 filled with the polymer solution 12 is reduced. The gap between the mm hole and the connection jig 8 and the gap between the capillary 1 and the connection jig 8 are sealed. As a result, the sample elution end 5 of the capillary is connected to the pressure-resistant block 10 with pressure. As the Z axis rises, gas tight syringe 11 and pressure block
10. When the load applied to the connection jig 8 increases and the load reaches 4 kg weight, the elevation of the Z axis is stopped. Here, the load is applied by raising the Z axis of the XZ stage 14, but the connecting jig 8 may be moved downward by another linear moving mechanism.

At this time, the polymer solution 12 in the gas-tight syringe 11 has a load of P = load / internal cross-sectional area of the syringe = 4 kg / 0.16 cm ² = 25 kg / cm ² = 2.5 × 10 6 N / m ² . High pressure is applied and the sample elution end 5 of the capillary 1 moves to the sample injection end 4
, The filling of the polymer solution 12 is started. If the Z-axis is kept stopped, the internal pressure decreases because the relative distance between the piston 13 of the gas tight syringe 11 and the pressure-resistant block 10 does not change with the filling of the polymer solution 12. Therefore, the signal of the load sensor is fed back so that the Z-axis is gradually raised so that the load applied to the piston 13 is always 4 kg. At this time, the filling speed of the polymer solution 12 is Q = (π · r4 · P) / (8 · μ · L) = 1.1 × 10, considering that the entire capillary is already filled with the polymer solution 12.
^-12 m ³ / sec. The internal volume of the capillary 1 is V = π
Since r ² · L = 1.5 × 10 ⁻⁹ m ³ , the filling time of the polymer solution corresponding to the internal volume is V / Q = 1364 sec = 23 min.
Therefore, the filling time of the polymer solution is set to 30 minutes with a margin.

After a lapse of 30 minutes, the Z axis of the XZ stage 14 is lowered, the internal pressure is relaxed, and the filling of the polymer solution is completed. Further, the Z-axis is lowered to separate the gas tight syringe 11 and the pressure-resistant block 10 from the capillary 1 and the connection jig 8. Subsequently, by controlling the XZ stage 16 to which the anode buffer solution tank 15 is fixed, the anode buffer solution solution 15 is moved directly below the capillary sample elution end 5 and the sample elution end 5 is moved to the buffer solution 17.
Soak in (1 × TBE).

FIG. 4 shows a configuration diagram of the sample injection device 6. The cathode buffer solution tank 18 and the sample tray 19 are set on the XZ stage 20. The sample tubes 21 to 24 are set on the sample tray 19, and different sample 25 to sample 28 are put in each sample tube. Each sample is a DNA sequencing sample using a different template DNA, and is a DNA sequencing sample preparation kit (ABI Pri
sm dRhodamine Terminator Cycle Sequencing Ready Re
Prepare with Action Kit With AmpliTaq DNA Polymerase (FS). The product is a group of DNA fragments that differ in length by one base, and each fragment is labeled with four different fluorophores corresponding to the terminal base type.

By moving the XZ stage 20, the cathode buffer solution tank 18 is moved immediately below the capillary sample injection end 4, and the sample injection end 4 is immersed in the buffer solution 29 (1 × TBE). The polymer solution filling device 7 causes the polymer solution 12 to elute the sample elution end of the capillary 1.
During the filling from 5 to the sample injection end 4, the sample injection end 4 of the capillary 1 is immersed in the buffer solution 29 all the time, and the polymer solution 12 eluted from the sample injection end 4 is placed in the cathode buffer solution 29. Elute.

After the filling of the polymer solution is completed and the sample elution end 5 is immersed in the anodic buffer solution 17, a high voltage of 7 kV is applied between the anodic buffer solution 17 and the cathodic buffer solution 10 for 10 minutes for preliminary Perform electrophoresis (electric field strength 200 V / cm). After preliminary electrophoresis, X
By moving the Z stage 20, the sample tube 21 on the sample tray 19 is moved directly below the capillary sample injection end 4, and the sample injection end 4 is immersed in the sample 25.

The cathode is immersed in the sample 25, and a high voltage of 1.4 kV is applied to the anode buffer 17 for 10 seconds to inject the sample (electric field intensity 40 V / cm). The XZ stage 20 is moved again, and the cathode buffer solution tank 18 is moved immediately below the capillary sample injection end 4, and the sample injection end 4 is immersed in the buffer solution 29 (1 × TBE). Electrophoresis is started by applying a high voltage of 7 kV between the anode buffer solution 17 and the cathode buffer solution 29 (electric field strength 200 V / cm).

At the same time as the start of the electrophoresis, the measurement by the fluorescence detector is started. Using a CCD camera, the emission fluorescence at the fluorescence measurement position of the capillary is continuously measured at an exposure time of 0.4 seconds and an interval of 0.7 seconds. Since the emission fluorescence is spectrally separated by a diffraction grating and collectively measured by a CCD camera, the luminescence of four phosphors in the sample can be measured independently and simultaneously. The base sequence of the template DNA of the sample 25 is determined by analyzing the time course of the four fluorescence intensities using a computer.

Two hours after the start of the electrophoresis, the electrophoresis and the fluorescence measurement are stopped, and the electrophoretic analysis of the sample 25 is completed. In order to perform the electrophoretic analysis of the sample 26 subsequently, the above-described polymer solution filling, preliminary electrophoresis, sample injection (here, the sample 26 is injected), electrophoresis and fluorescence measurement,
Perform computer analysis cycle. By automatically repeating this cycle under computer control,
Electrophoretic analysis of any number of samples can be performed unattended. In the present embodiment, four types of sample tubes are arranged on the sample tray 19, but continuous measurement can be performed by the same algorithm regardless of the number of sample tubes.

If it is necessary to clean the inner surface of the capillary between one electrophoresis measurement and the next electrophoresis measurement, another combination of an XZ stage, a gas tight syringe and a pressure-resistant block must be installed in the polymer filling device. Prepare one. The inside of the 1 mm diameter hole of the gas tight syringe and pressure block is filled with the cleaning liquid. When filling polymer solution
If the XZ stage is operated in the same manner as the moving means of the XZ stage 14, the cleaning liquid in the gas tight syringe is filled in the capillary, and the cleaning is performed.

In the present embodiment, the connecting jig 8 has a conical shape, and the pressure-resistant block 10 is formed with a conical hole that is compatible with the conical hole. The effect is obtained. The material of the connection jig is desirably a material having a large elastic modulus and a high compressibility as compared with the material of the pressure-resistant block.

FIG. 5 is a diagram showing an example of this, and corresponds to FIG. FIG. 5A is a view before the collision between the connection jig 30 and the pressure-resistant block 31, and FIG. 5B is a view after the collision.
The connection jig 30 is made of silicon and has a diameter of 10 mm and a height of 5 m.
It has a cylindrical shape of m and a hole with a diameter of 0.5 mm in the center. The pressure-resistant block 31 is made of acrylic material, has a bottom diameter of 20 mm,
Drill a 1 mm diameter hole in the center with a 40 mm high cylinder. After the sample elution end 5 of the capillary 1 is made to penetrate the hole of the connection jig 30, it is inserted into the hole of the pressure-resistant block 31. The upper surface of the connection jig 30 is held by the holder 9 as in the case of FIG.

When the relative distance between the connection jig 30 and the pressure-resistant block 31 is reduced by the linear drive mechanism, the connection jig 30 collides with the pressure-resistant block 31 and is deformed. As a result, the connection jig 30
Has a small height, a large bottom surface, and a small hole in the center. Thereby, the gap between the capillary 1 and the hole of the connection jig 30 is sealed, and the gap between the connection jig 30 and the hole of the pressure-resistant block 31 is also sealed. Therefore, a pressure-resistant connection between the capillary 1 and the pressure-resistant block 31 is formed.

This pressure-resistant connection becomes stronger as the force for reducing the hole formed in the center due to the deformation of the connection jig 30 becomes larger. In FIG. 5, the connecting jig 30 is expanded in the direction in which the bottom surface is enlarged by deformation. However, if the deformation in this direction can be suppressed, the force for reducing the hole formed in the center can be increased.

FIG. 6 shows a breakdown voltage block 32 for this purpose.
FIG. 5 is a diagram showing before and after a collision between the connector jig and the connection jig 30. Since a recess is formed in the upper surface of the pressure-resistant block 32 to just fit the connection jig 30, deformation in the direction in which the bottom surface is increased is suppressed, and the hole of the connection jig 30 is easily reduced. Therefore, the capillary 1 can be connected to the pressure-resistant block 32 with high pressure.

Example 2 The polymer solution filling apparatus of the present invention is applied to a sheath flow type electrophoresis analyzer using 48 capillary arrays.

The electrophoresis buffer was 1 × TBE (89 mM tris (hy
droxymethyl) aminomethane, 89 mMboric acid, 2 mM e
thylenediaminetetraacetic acid (EDTA)). 1.5% molecular weight polymer solution in electrophoretic separation medium
8,000,000 PEO (poly (ethylene oxide)) and 1.4%
Of PEO with a molecular weight of 600,000 dissolved in 1 × TBE containing 3.5 M urea (Aldrich, USA)
Use Sigma). The viscosity of this polymer solution is μ = 1200
A CP = 1.2 N · sec / m 2 (Analytical Chemistry 6
7, 1913 (1995)).

Outer diameter R = 0.200 mm, inner diameter r = 0.0075 mm, overall length
Forty-eight capillaries of L = 550 mm (Polymicro) are arranged on the same plane so as to satisfy the following conditions. A range from one end of each capillary to 100 mm is parallel to each other, and arranged at an arrangement interval of 3 mm and a total width of 141 mm. At this time, the ends are aligned so that the line connecting the ends of the capillaries is perpendicular to the axis of each capillary. On the other hand, the range up to 100 mm from the opposite end of each capillary is
It is arranged in a total width of 18.8 mm with 0.2 mm. At this time, the ends of the capillaries do not have to be aligned, and priority is given to the whole of each capillary being on the same plane.

Adhesive tapes are stuck to several places on the capillary array so that the arrangement interval at both ends is maintained. Cut all capillaries perpendicular to the axis of each capillary at the center of the portion where the array interval of the capillary array is 0.2 mm, and align the tips. At this time, the total length of the capillary arranged at the center of the capillary array is set to L = 500 mm. The end on the side where the array interval of the capillary array is 3 mm is the sample injection end, and the end on the side where the array interval is 0.2 mm is the sample elution end. The total length of the capillary becomes slightly longer from the center to both ends in the capillary array. The total length of the capillaries arranged at both ends is 500.8 mm, while the total length of the capillaries arranged at the center is 500 mm, and the difference is 1% or less. Therefore, all the 48 capillaries can be analyzed under almost the same electrophoresis conditions.

The sample elution end of the capillary array is placed in a sheath flow cell made of quartz glass. Inside the sheath flow cell, the electrophoresis buffer flows at a speed of about 0.1 mm / sec in the direction of electrophoresis of the capillary (the direction from the sample injection end to the sample elution end). The sheath flow cell doubles as an anode buffer tank during electrophoresis. The direction of the sample elution end and the sheath flow may be vertical or horizontal, but in this case, the direction is vertically downward. Inside the sheath flow cell, a laser is irradiated at a position where no capillary exists, downstream of the sample elution end from the sample elution end, in a direction perpendicular to the capillary axis and parallel to the plane where the capillaries are arranged ( JP-A-6-138037). The laser uses 488 nm and 514.5 nm argon ion lasers.

The sample components electrophoresed in each capillary from the sample injection end to the sample elution end are eluted from the sample elution end into the buffer inside the sheath flow cell, and flow downstream of the buffer flow. When the flowed sample component traverses the laser beam, the fluorescent substance labeled on the sample component is excited and emits fluorescence. The sample components eluted from the different capillaries do not mix with each other in the buffer, since the buffer flow forms a laminar flow.

The emitted fluorescent light is received as a two-dimensional image by the CCD camera from a direction perpendicular to the plane on which the capillaries are arranged. By placing an image splitting system immediately before the CCD camera, it is possible to simultaneously detect light emission from four different phosphors. The image dividing system divides an image into four in the axial direction of the capillary so that each image passes through four different types of optical filters and is formed (JP-A-2-269936). With the above-described fluorescence detection device, the sample components eluted from each capillary are detected independently and collectively by fluorescence.

On the other hand, the sample injection end 52 of the 48 capillaries 51
Are installed in 48 connection jigs 53 as shown in FIG. The direction of the sample injection end 52 may be a horizontal direction or a vertical direction, but here, it is directed vertically downward. The connection jig 53 is made of Teflon, has a conical shape with a bottom surface diameter of 2 mm and a height of 3 mm, and has a 0.22 mm hole at the center thereof. A 0.5 mm diameter taper is formed on the bottom side of the hole of the connection jig. The holder 54 is made of aluminum and has a strip shape, and 48 holes of 0.5 mm in diameter penetrate the straight line at an arrangement interval of 3 mm in the long side direction. The 48 connection jigs 53 are arranged at intervals so that the holes of the connection jig and the holes of the holder are coaxial.
It is held on a holder at 3 mm. 48 capillaries 51
The sample injection end 52 is passed through each hole of the holder 54 and the connection jig 53 while keeping the arrangement interval. The holder 54 is connected to and fixed to a Z stage movable in the vertical direction. The Z stage has a built-in load sensor that can measure the load applied in the Z direction.

Below the sample injection end 52 of the capillary array 55, a polymer solution filling unit 56, a cathode buffer solution tank 57, and a sample tank
There is an YZ stage 62 in which 58 to 61 are held in order. The Z axis is a vertical movement of each unit, and each unit can be moved closer to or farther from the sample injection end 52. The Y axis moves in the direction perpendicular to the plane of the paper in Fig. 7, and the desired unit is moved to the sample injection end.
You can move directly under 52.

FIG. 8 is a view of the YZ stage 62 viewed from above vertically, and the vertical direction of the drawing is the Y-axis direction. The polymer solution filling unit 56 includes a pressure-resistant block 63, a gas tight syringe 64,
It consists of a Y stage 65. The inside of the pressure-resistant block 63 and the gas tight syringe 64 is a polymer solution to be filled in the capillary 51.
66 is satisfied. The Y stage 65 has a built-in load sensor, and can measure a load applied in the Y direction. On the upper surface of the pressure-resistant block 63, there are 48 capillaries.
48 tapered holes are arranged linearly at an interval of 3 mm for pressure-resistant connection with 51 sample injection ends 52 and 48 connection jigs 53
And is in communication with the polymer solution 66 inside the pressure-resistant block 63. The gas tight syringe 64 is pressure-tightly connected to the pressure-resistant block 63 at the central portion, and the polymer solutions 66 inside communicate with each other.

The Y stage 65 is fixed to the piston 67 of the gas tight syringe 64, and can move the piston 67 by moving the piston 67 up and down on the plane of FIG. The cathode buffer solution tank 57 has a width of 150 mm, and can simultaneously immerse the sample injection ends 52 of the 48 capillaries 51. Sample tanks 57-61
Four are mounted, and each sample tank holds 48 different samples. 48 holes of 1 mm in diameter are arranged in a straight line on each sample tank
Drilled at 3 mm, each hole holds 48 samples.
Each sample was prepared using a different template DN in the same manner as in Example 1.
A DNA sequencing sample prepared from A.

By moving the Y axis of the YZ stage 62,
The 48 holes of the pressure-resistant block 63 are arranged directly below the 48 sample injection ends of the capillary array. The Z-axis of the YZ stage 62 is raised, and the rise of the Z-axis is stopped immediately before the connection jig 53 comes into contact with the pressure-resistant block 63. FIG. 9 shows an enlarged view of the contact portion between the two at this time. Each sample injection end 52 is guided to the tapered hole of the pressure-resistant block 63,
63 is inserted into the polymer solution 66 inside. Next, the Z stage to which the connection jig 53 and the holder 54 are fixed is lowered, and when the load applied to the entire pressure-resistant block 63 reaches 10 kg weight, the lowering is stopped and the state is maintained. At this time, a pressure-resistant connection is formed between the sample injection end 52, the connecting jig 53, and the tapered hole of the pressure-resistant block 63 at each position by a mechanism similar to that of FIG.

The Y stage 65 connected to the piston 67 of the gas tight syringe 64 is moved, and the gas tight syringe 6 is moved.
4 and the pressure of the polymer solution 66 inside the pressure-resistant block 63 is increased. When the load applied to the piston 67 by the Y stage 65 reaches 4 kg weight, the movement of the Y stage 65 is stopped. At this time, the polymer solution 66 inside the gas tight syringe 64 contains P
= Load / Syringe internal cross-sectional area = 4 kg weight / 0.16 cm ² = 25 kg
Weight / High pressure ^{cm 2 = 2.5 × 10 6 N} / m 2 is applied, filling the polymer solution 66 is started toward the sample injection end of the sample elution end 52 of 48 capillaries 51.

When the Y stage 65 is kept stopped, the internal distance decreases because the relative distance between the piston 67 of the gas tight syringe 64 and the pressure-resistant block 63 does not change with the filling of the polymer solution 66. Therefore, the signal of the load sensor is fed back so that the Y stage 65 is moved little by little so that the load applied to the piston 67 always becomes 4 kg.

At this time, the polymer solution inside each capillary 51
The filling rate of 66 is Q = (π · r4 · P) /, considering the state that the entire capillary is already filled with the polymer solution 66.
(8 μL) = 3.2 × 10 ⁻¹² m ³ / sec. Since the internal volume of each capillary is V = π · r ² · L = 2.2 × 10 ⁻⁹ m ³ , the polymer solution filling time corresponding to the internal volume is V / Q = 688 s
ec = 11 min.

Therefore, the filling time of the polymer solution is set to 15 minutes with a margin. During the filling of the polymer solution 66, the polymer solution is discharged from the sample eluting end of each capillary 51 into the buffer solution inside the sheath flow cell, but this is flowed into the buffer solution stream and discarded. There is no stagnation inside and the fluorescence measurement is not hindered. After 15 minutes,
The Y stage 65 is moved in the opposite direction to reduce the internal pressure of the gas tight syringe and the pressure-resistant block, thereby completing the filling of the polymer solution.

The Z stage to which the connection jig 53 and the holding jig 54 are fixed is lifted to reduce the load applied to the pressure-resistant block 63 and return to the state shown in FIG.
The pressure-resistant connection formed between the sample injection end 52, the connection jig 53, and the tapered hole of the pressure-resistant block 63 is released. Next, the Z axis of the YZ stage 62 is lowered, and the pressure-resistant block 63 is separated from the sample injection end 52 of each capillary 51 and the connection jig 53.

By moving the Y axis of the YZ stage 62,
The cathode buffer solution tank 57 is provided immediately below the sample injection end 52 of the capillary array 55. Raise the Z-axis of the YZ stage 62 so that all the sample injection ends 52 of the 48 capillaries 51 have buffer solution.
Stop raising the Z axis while immersed in 68. FIG. 10 shows an enlarged view of this state. The connection jigs 53 installed in the capillaries 51 are not immersed in the buffer solution 68. Next, a high voltage of 10 kV is applied between the anode buffer solution and the cathode buffer solution 68 in the sheath flow cell for 10 minutes to perform preliminary electrophoresis (electric field intensity: 200 V / cm). After the preliminary electrophoresis, the Z axis of the YZ stage 62 is lowered, and the cathode buffer tank 57 is separated from the sample injection end 52 of the capillary array 55.

By moving the Y-axis of the YZ stage 62, the sample tank 58 is positioned directly below the sample injection end 52 of the capillary array 55. The Z-axis of the YZ stage 62 is raised, and the Z-axis is raised with the sample injection ends 52 of the 48 capillaries 51 inserted into the 48 types of samples 69 held in the 48 holes on the sample tank 58. Stop. An enlarged view of this state is shown in FIG. In 48 kinds of samples 69 held in 48 holes on the sample tank 58,
A platinum electrode 70 is inserted from the opposite direction to the insertion of the capillary 51. Using these electrodes as cathodes, a high voltage of 2 kV is applied for 10 seconds between the 48 kinds of sample solutions 69 and the anode buffer to perform sample injection (electric field strength of 40 V / c).
m). Forty-eight kinds of samples 69 are collectively injected into 48 capillaries 51, respectively. The Z axis of the YZ stage 62 is lowered, and the sample tank 58 is separated from the sample injection end 52 of the capillary array 55.

By moving the Y axis of the YZ stage 62,
The cathode buffer tank 57 is again positioned immediately below the sample injection end 52 of the capillary array 55. The Z-axis of the YZ stage 62 is raised so that all the sample injection ends 52 of the 48 capillaries 51 are immersed in the buffer solution 68 as in FIG. A high voltage of 10 kV is applied between the anode buffer and the cathode buffer 68 to start electrophoresis (electric field intensity 200 V / cm).

At the same time as the start of the electrophoresis, the measurement by the fluorescence detector is started. Using a CCD camera, the emission fluorescence from 48 fluorescence measurement positions in the sheath flow portion is continuously measured at a time with an exposure time of 0.4 seconds and an interval of 0.7 seconds. Emission fluorescence is measured by a CCD camera using an image division system, so the emission of four phosphors in a sample can be measured independently and simultaneously. The nucleotide sequences of 48 template DNAs are determined by analyzing the time course of the four fluorescence intensities using a computer.

Two hours after the start of the electrophoresis, the electrophoresis and the fluorescence measurement are stopped, and the electrophoretic analysis of the 48 kinds of samples in the sample tank 58 is completed. To perform the electrophoretic analysis of the 48 kinds of samples on the sample tank 59 continuously, the above-described polymer solution filling, preliminary electrophoresis, sample injection (here, 48 kinds of samples on the sample tank 59 are injected), electrophoresis And cycle of fluorescence measurement and computer analysis. By automatically repeating this cycle under computer control, electrophoresis analysis of an arbitrary number of sample tanks can be performed unattended. In the present embodiment, the four types of sample tanks 57 are mounted on the YZ stage 62.
Although ~ 61 are arranged, continuous measurement can be performed by the same algorithm regardless of the number of sample vessels.

In this embodiment, a plurality of capillaries and one
Although a plurality of withstand voltage blocks correspond, a plurality of withstand voltage blocks may correspond.

In the present embodiment, the sample injection ends of a plurality of capillaries, the connecting jig, and the tapered holes of the pressure-resistant block are arranged linearly at the same arrangement interval, but these are arranged two-dimensionally. May be. In this case, it is desirable to arrange them regularly in a grid pattern.

In this embodiment, a gas tight syringe is used as a means for increasing the pressure inside the pressure-resistant block. However, other types of pumps, such as a pump for liquid chromatography, may be used.

[0079]

The solution filling apparatus proposed in the present invention can be combined with a sheath flow type capillary array electrophoresis apparatus which is a high-sensitivity, high-throughput DNA analysis apparatus, thereby automatically performing a plurality of electrophoresis analyses. The operation can be repeated repeatedly, so that the labor required for large-scale analysis can be significantly reduced and the throughput can be further increased.
In addition, this solution filling device fills multiple types of solutions,
Since both ends of the capillary can be immersed in an arbitrary buffer, a wider range of electrophoresis conditions can be set.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a single capillary electrophoresis apparatus for on-column fluorescence detection.

FIG. 2 is a sectional view showing a configuration of a polymer solution filling apparatus.

FIG. 3 is a sectional view showing a pressure-resistant connection between a capillary and a pressure-resistant block by a connection jig.

FIG. 4 is a sectional view showing a configuration of a sample injection device.

FIG. 5 is a cross-sectional view showing a pressure-resistant connection between a capillary and a pressure-resistant block by a connection jig.

FIG. 6 is a cross-sectional view showing a pressure-resistant connection between a capillary and a pressure-resistant block by a connection jig.

FIG. 7 is a schematic cross-sectional view showing a configuration near a sample injection end of a capillary array.

FIG. 8 is a plan view showing a polymer solution filling and sample injection device for a capillary array.

FIG. 9 is an enlarged cross-sectional view near the sample injection end when the capillary array is filled with a polymer solution.

FIG. 10 is an enlarged cross-sectional view near the sample injection end during electrophoresis of a capillary array.

FIG. 11 is an enlarged cross-sectional view near the sample injection end when a sample is injected into the capillary array.

[Explanation of symbols]

1 ... capillary, 2 ... fluorescence detector capillary holder,
3 ... temperature control plate, 4 ... sample injection end, 5 ... sample elution end, 6 ... sample injection device, 7 ... polymer solution filling device, 8 ... connection jig, 9 ... holding fixture, 10 ... pressure resistant block, 11 ... gas Tight syringe, 12
... Polymer solution, 13 ... Piston, 14 ... XZ stage, 15 ... Anode buffer solution, 16 ... XZ stage, 17 ... Anode buffer solution, 18 ... Cathode buffer solution, 19 ... Sample tray, 20 ... XZ stage, 21 ... Sample tube, 22… Sample tube, 23… Sample tube, 24
… Sample tube, 25… sample, 256… sample, 27… sample, 28
... sample, 29 ... cathode buffer, 30 ... connection jig, 31 ... pressure-resistant block, 32 ... pressure-resistant block, 51 ... capillary, 52 ... sample injection end, 53 ... connection jig, 54 ... holder, 55 ... capillary array , 56… Polymer solution filling unit, 57… Cathode buffer tank,
58… Sample tank, 59… Sample tank, 60… Sample tank, 61… Sample tank, 62
... YZ stage, 63 ... pressure block, 64 ... gas tight syringe, 65 ... Y stage, 66 ... polymer solution, 67 ... piston, 68 ... cathode buffer, 69 ... sample, 70 ... platinum electrode.

Claims (6)

[Claims] 1. A solution filling apparatus for filling a capillary with a solution, a pressurizing means for applying pressure to an internal solution, a capillary filled with the solution, and a solution which becomes a closed container when the pressurizing means and the capillary are connected. A solution tank is filled with the solution, a first hole larger than at least the outer diameter of the capillary is formed in a capillary connection portion of the solution tank, and when the capillary is connected to the solution tank, one end of the capillary is in the first position. Immersed in the solution inside the solution tank through the hole of
The solution inside the pressurizing means and the solution inside the solution tank are connected by connecting the pressurizing means to the solution tank, and the connection between the pressurizing means and the solution tank is connected when a high pressure is applied to the solution inside the solution tank. The connection between the capillary and the solution tank is a pressure-resistant sealed connection in which the solution does not leak from the connection part to the outside when a high pressure is applied to the solution inside the solution tank. The pressure tight sealing connection and the pressure tight sealing disconnection of the solution tank and the pressure tight sealing connection are mechanically performed, and the pressure tight sealing connection means of the capillary and the solution tank has a compressible connecting jig at least partially larger than the minimum portion of the first hole. Then, a second hole larger than at least the outer diameter of the capillary is formed in the connection jig, and one end of the capillary penetrates the second hole and then penetrates the first hole and is immersed in the solution in the solution tank. Connect the connection jig to the solution tank Is a means for compressively deforming the connection jig by pressing against the gap, sealing the gap between the capillary and the second hole, and simultaneously sealing the first hole of the connection portion of the solution tank. Deformation means is a means for reducing the relative distance between the connection jig and the connection part of the solution tank by a linear drive mechanism, colliding the connection part of the connection jig and the connection part of the solution tank, and applying a load to the connection jig. Solution, wherein the pressure-tight closed connection releasing means of the solution tank is a means for increasing the relative distance between the connection jig and the connection part of the solution tank by a linear drive mechanism and removing the load applied to the connection jig. Filling device. 2. A solution filling apparatus for filling N-capillaries with a solution in which N is an arbitrary natural number, a pressurizing means for applying pressure to an internal solution, an N-capillary filled with the solution, and a pressurizing means. And a solution tank that becomes a closed container by connecting the N capillaries, the solution tank is filled with the solution, and the N connections of the solution tank to which the N capillaries connect are at least outside the capillaries. First larger than diameter
When N capillaries are opened and N capillaries are connected to the solution tank, one end of each of the N capillaries is immersed in the solution inside the solution tank through each of the N first holes, and the pressurizing means is provided. By connecting to the solution tank, the solution inside the pressurizing means and the solution inside the solution tank are connected, and the connection between the pressurizing means and the solution tank is connected to the outside when a high pressure is applied to the solution inside the solution tank. It is a pressure tight connection that does not leak the solution, and the connection between the N capillaries and the solution tank is a pressure tight connection that does not leak the solution from the N connections to the outside when high pressure is applied to the solution inside the solution tank. The pressure-tight sealing connection between the N capillaries and the solution tank and the pressure-tight sealing disconnection are mechanically performed, and the pressure-tight sealing connection means between the N capillaries and the solution tank is at least partially connected to the first.
Has a number N of compressible connection jigs larger than the minimum part of the holes, each of the N connection jigs has at least a second hole larger than the outer diameter of the capillary, and one end of the N capillaries is formed. After penetrating each of the N second holes, each is penetrated through the N first holes and dipped in the solution in the solution tank, and the N connection jigs are pushed into the N connection portions of the solution tank. The N connection jigs are compressed and deformed by contact, and the gap between the N capillaries and the second holes respectively penetrated is sealed, and at the same time, the first holes of the N connection portions of the solution tank are closed. The sealing means, the compression deformation means of the N connection jigs, the linear drive mechanism reduces the relative distance between the N connection jigs and the N connection portions of the solution tank, and the N connection jigs This is a means for applying a load to the N connection jigs by colliding the N connection parts of the solution tank with the N connection jigs. The closed connection release means is means for increasing the relative distance between the N connection jigs and the N connection portions of the solution tank by a linear drive mechanism and removing the load applied to the N connection jigs. A solution filling device. 3. The solution filling apparatus according to claim 1, wherein the connecting jig has a cylindrical shape, and a second hole is formed in a center axis of the connecting jig. A solution filling device, wherein at least a part of the bottom surface facing the first hole collides with a connecting portion of the solution tank to compress and deform the connecting jig. 4. The solution filling apparatus according to claim 1, wherein the connecting jig has a cylindrical shape, a second hole is formed in a center axis of the connecting jig, and a vertex of the connecting jig is formed in the solution tank. The first hole of the connecting portion is opposed to the first hole, and the first hole is formed with a taper that matches the cylindrical shape of the connecting jig, and at least a part of the side surface of the connecting jig has a tapered portion of the first hole. A solution filling device, wherein the connection jig is compressed and deformed by collision with the solution. 5. A solution filling apparatus according to claim 1, wherein a plurality of solution tanks are provided, each solution tank is filled with the same solution or a different solution, and each capillary and each solution tank are pressure-tightly sealed and connected. A solution filling device, which can be released and each capillary can be filled with each solution. 6. The solution filling apparatus according to claim 1, wherein each capillary is used for electrophoresis, and a direction in which each solution is filled in each capillary and a direction in which a sample introduced into each capillary migrates. A solution filling apparatus characterized by the above-mentioned.