JP3838798B2 - Sample plate and multicapillary electrophoresis device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
In the present invention, a plurality of capillary columns are arranged, a plurality of samples are injected into the capillary columns one by one, and the capillary is electrophoresed simultaneously in all the capillary columns, and the capillary is irradiated with light in the multi-capillary array electrophoresis unit And a multi-capillary electrophoresis apparatus having an optical measurement unit for measuring absorbance and fluorescence from the sample in the irradiated portion, and a sample plate used when introducing the sample into the plurality of capillary columns. Is.
Such a multi-capillary electrophoresis apparatus is used for protein separation and DNA base sequence determination. In a multicapillary electrophoresis apparatus for determining the base sequence of DNA, a DNA fragment (fragment) sample with a primer or terminator labeled with a fluorescent substance is electrophoresed using a Sanger reaction, and fluorescence from the DNA fragment sample is migrated during the electrophoresis. The base sequence is determined by detection.
[0002]
[Prior art]
In order to determine the base sequence of a DNA having a long base sequence such as the human genome, a DNA sequencer having high sensitivity, high speed, and large throughput is required. As one of the methods, a multicapillary DNA sequencer in which a plurality of capillary columns packed with gels are arranged instead of using a flat slab gel has been proposed. Capillary columns are not only easier to handle and inject samples than slab gels, but also can be migrated at high speed and detected with high sensitivity. In other words, if a high voltage is applied with a slab gel, problems such as band expansion or temperature gradients occur due to the effect of Joule heat, but such problems are few with capillary columns. Even if it is made, it is possible to perform highly sensitive detection with little band spread.
[0003]
Samples are introduced into a capillary column in capillary electrophoresis by a method using pressure or an electrophoretic method in which a voltage is applied. Among them, a method for introducing a sample by electrophoresis is widely and generally adopted from the viewpoint of simplicity of apparatus configuration, ease of operation, and good controllability of parameters.
When introducing a sample by electrophoresis, it is necessary to immerse one end of the capillary column in the prepared sample, immerse the other end in a buffer solution, and immerse an electrode such as a platinum wire in the vicinity of the end of the capillary column in the sample. is there.
[0004]
[Problems to be solved by the invention]
When the electrode is held near the end of each capillary column when introducing an electrophoretic sample, in the case of a multi-capillary electrophoresis apparatus that arranges multiple capillary columns in an array and simultaneously performs electrophoresis, sample introduction This makes the electrode structure complicated.
In addition, the capillary column end must be immersed in the sample in the sample injection container and injected, and then the capillary column end must be transferred to a reservoir containing the buffer solution for electrophoresis. It would be advantageous if the operation up to and including this would be troublesome and could be automated.
[0005]
A first object of the present invention is to provide a sample plate for sample introduction having a simple electrode structure suitable for a multi-capillary electrophoresis apparatus.
A second object of the present invention is to provide a multi-capillary electrophoresis apparatus using such a sample plate and capable of automating from sample injection to electrophoresis in a large number of capillary columns.
[0006]
[Means for Solving the Problems]
The sample plate of the present invention includes a disposable insulating resin base plate in which one or a plurality of holes are formed as wells, the bottoms of the wells are opened, and each protrudes to the same height, and the base plate includes Conductive metal for fixing the base plate by forming recesses having dimensions smaller than the dimensions of the wells at positions corresponding to the two-dimensional array of wells formed in an array, and pressing the bottom of the wells into the recesses. An electrode plate.
[0007]
The multi-capillary electrophoresis apparatus of the present invention includes a multi-capillary array electrophoresis unit in which a plurality of capillary columns are arranged, a plurality of samples are injected into the capillary columns one by one, and electrophoresed simultaneously on all the capillary columns, and a multi-capillary array electrophoresis In a multi-capillary electrophoresis apparatus equipped with an optical measurement unit that irradiates the capillary with light and measures the absorbance of the irradiated part of the sample and the fluorescence from the sample, the sample injection side of the multi-capillary array migration unit The two-dimensionally arranged capillary column end is fixed downward, and the sample plate in which the wells containing the sample are two-dimensionally arranged below the capillary column end corresponding to the arrangement of the capillary column end. And buffer solution for electrophoresis Is an electrophoretic reservoir voltage to all the capillary is applied is arranged, the moving mechanism is provided with one of the sample plate and the electrophoresis reservoir into contact with the capillary end switches.
[0008]
The well of the base plate is press-fitted and fixed in the recess of the electrode plate to form a space for the sample, and the sample is put in each well.
Since such a base plate is a disposable type, contamination can be prevented. The configuration may be either n well / row or n well × m row for each well. Considering that the molding material is disposable, general-purpose engineering plastics having chemical resistance and good moldability, such as polypropylene and polyethylene, are suitable.
[0009]
At the time of sample injection into the capillary column, the sample is put in each well of the sample plate, the sample plate is moved by the moving mechanism, and one end of each capillary column is immersed in the sample in the well. A voltage is applied between the electrode plate of the sample plate and the other end of the capillary column to inject the sample into the capillary electrophoretically. After sample injection, the sample plate is removed from contact with one end of the capillary column by the moving mechanism, and one end of the capillary column is immersed in the buffer solution of the electrophoresis reservoir. Then, electrophoresis is performed by applying a voltage between the buffer solutions in the reservoirs at both ends of the capillary column. As a result, operations from sample injection to capillary column to electrophoresis are automatically performed.
[0010]
In addition to the case where a sample already processed is put in the well of the sample plate, after processing the sample by PCR (Polymerase Chain Reaction) using the well of the sample plate, the capillary end is attached to the sample in the well. It can also be used to insert and perform sample injection. The PCR method is a method for greatly amplifying only a desired portion of DNA. In the PCR method, a primer is added to the sample DNA, the temperature is raised to dissociate the double-stranded DNA into single strands, then the temperature is lowered to bind the primer to the DNA strand, and the temperature is raised slightly to synthesize the DNA. Increase the temperature further to make it single-stranded. In this way, by repeating the operation of changing the temperature up and down, a predetermined portion of DNA is amplified and synthesized in large quantities.
[0011]
【Example】
FIG. 1A is a perspective view showing a base plate of one embodiment. (B) is sectional drawing in the ZZ line position in (A).
The base plate 101 is entirely formed of a thin plastic, and the wells 102 having holes opened at the bottom are arranged at regular intervals on the flat surface thereof. The inner diameter of the well 102 is narrow at the bottom contacting the electrode plate and thick at the top where the capillary is inserted. The size of the bottom opening is 0.7 mm to 2 mm, and the size of the top opening is 4 mm to 4.5 mm. Thus, when the bottom opening is closed by the electrode plate 104 described later and the sample is accommodated, the liquid level can be increased even if the sample amount is small, and the capillary can be easily guided to the sample in the well 102. it can.
[0012]
FIG. 2A is a perspective view showing a sample plate of one embodiment. (B) is sectional drawing in the YY line position in (A).
The electrode plate 104 is made of a conductive material having high mechanical strength, such as stainless steel, and recesses 105 arranged in the vertical and horizontal directions are formed on the planar surface. A plurality of base plates 101 are arranged on the surface of the electrode plate 104. The array of the depressions 105 corresponds to the two-dimensional array of wells 102 formed by arranging the base plate 101 on the surface of the electrode plate 104. The size of the recess 105 is slightly smaller than the outer dimension of the bottom of the well 102 in order to press-fit the bottom of the well 102 and fix the base plate 101. At the bottom of the electrode plate 104, a migration high voltage line connection hole 107 is formed for connection to a high voltage power source through a plug 106 installed in the electrophoresis apparatus main body.
[0013]
A well guide 103 guides the well 102 to the recess 105 of the electrode plate 104. The well guide is fixed to the surface of the electrode plate 104, and a through hole is formed at a position corresponding to the recess 105. The well guide 103 may be a conductor or an insulator, and any material may be used.
The base plate 101, the well guide 103, and the electrode plate 104 constitute a sample plate 100.
[0014]
After use, the electrode plate 104 is removed from the base plate 101. The base plate 101 is discarded after one use, but the electrode plate 104 and the well guide 103 are repeatedly used. Wash the electrode plate 104 before reusing it. In particular, since the recess 105 has a portion that comes into contact with the sample, it must be washed before use.
[0015]
FIG. 3 schematically shows a multi-capillary electrophoresis apparatus that performs sample injection using this sample plate.
The multi-capillary electrophoresis device is a gel electrophoresis device in which polyacrylamide gel, linear acrylamide gel, polyethylene oxide (PEO) gel, etc. are packed as separation media in the capillary column. Free electrophoresis in the capillary column without filling the gel. However, the sample plate of the present invention can be applied to any electrophoresis apparatus using a plurality of capillary columns.
[0016]
Buffer liquids 112 and 122 are accommodated in a pair of reservoirs 110 and 120, respectively, and electrodes 130 and 132 are provided in both buffer liquids, respectively.
The electrode plate 104 of the sample plate 100 shown in FIG. 2 is installed in the electrophoresis apparatus body, and a plug 106 is inserted into the electrophoresis high voltage line connection hole 107 and connected to a high voltage wiring cable. Each well of the base plate 101 is inserted into a through hole of the well guide 103 and further press-fitted into a recess of the electrode plate 104, so that the base plate 101 is fixed to the electrode plate 104. Thereafter, a sample is put in each well of the base plate 101. If necessary, the sample is gently defoamed so that the sample contacts the electrode plate 104.
[0017]
The reservoir 110 and the sample plate 100 are connected so as to be switched by a high voltage switching unit 136 so that wiring can be switched, and a high voltage power source for electrophoresis is connected between the high voltage switching unit 136 and the electrode 132 provided in the other reservoir 120. The voltage for sample injection and electrophoresis is applied.
[0018]
At the time of sample injection, one end portion 2 a of the capillary array 2 is inserted into each well of the sample plate 100, and after sample injection, the reservoir 110 is switched and the one end portion 2 a is immersed in the buffer solution 112. The other end 2b of the capillary array 2 is immersed in the buffer solution 122 of the other reservoir 120, and the other end is irradiated with excitation light or measurement light from the optical measurement unit 10 that detects the sample by absorbance or fluorescence. And a detected portion 2c for measuring fluorescence.
[0019]
The capillary array 2 has a two-dimensional arrangement corresponding to the arrangement of the wells 102 of the sample plate 100 on one end side 2a, and the capillary column is arranged in a line in the detected portion 2c, and measurement is performed from a direction perpendicular to the arrangement surface of the capillary column. Light or excitation light is irradiated.
The sample plate 100 and the reservoir 110 are switched by a moving mechanism (not shown in FIG. 3) so as to selectively come into contact with the capillary end 2a.
[0020]
At the time of sample injection, the capillary column end 2 a is immersed one by one in the sample in the well of the sample plate 100, and the other end of the capillary column is immersed in the buffer solution 122 of the reservoir 120 together. Then, a high voltage is simultaneously applied to all the wells 102 through the electrode plate 104 to inject the sample into the capillary column.
[0021]
After the sample injection, the high voltage application is temporarily stopped, and the sample plate 100 and the reservoir 110 are moved by the moving mechanism, so that the capillary end 2a on the sample side is immersed in the buffer solution 112 of the reservoir 110. Thereafter, electrophoretic separation is performed by applying a high voltage between the reservoirs 110 and 120.
[0022]
In the method of connecting the high voltage line to the electrode plate 104, a plate spring or the like connected to the high voltage power source 136 is provided at a position where the sample plate 100 is fixed, and the plate spring and the electrode plate 104 come into contact when the sample plate 100 is fixed. You may make it do.
The moving mechanism may move the sample plate 100 and the reservoir 110 in a horizontal plane and move the capillary end 2a in the vertical direction.
The number of wells in the sample plate 100 can be arbitrarily set according to the number of capillary arrays.
[0023]
【The invention's effect】
The sample plate of the present invention has a disposable insulating resin base plate in which one or a plurality of holes are formed as wells, the bottoms of the wells are opened, and each protrudes to the same height, and the base plate is arranged. Conductive metal electrode plates for fixing the base plate by forming recesses having dimensions smaller than the well dimensions at positions corresponding to the two-dimensional array of wells to be formed, and pressing the bottom of the wells into the recesses. When this sample plate is used, it is possible to apply a voltage to a plurality of wells without taking a complicated electrode wiring structure. In particular, sample injection into a capillary column in an electrophoresis apparatus using a large number of multi-capillary arrays is facilitated.
The multi-capillary electrophoresis apparatus of the present invention uses this sample plate for sample injection, and has a moving mechanism for switching either the sample plate or the electrophoresis reservoir to contact the end of the capillary column. The injection and electrophoresis operations can be performed automatically.
[Brief description of the drawings]
1A and 1B show an embodiment of a base plate, in which FIG. 1A is a schematic perspective view, and FIG. 1B is a cross-sectional view taken along the line ZZ in FIG.
2A and 2B show an embodiment of a base plate, in which FIG. 2A is a schematic perspective view, and FIG. 2B is a cross-sectional view taken along the line YY in FIG.
FIG. 3 is a schematic perspective view showing an embodiment of a multi-capillary electrophoresis apparatus that performs sample injection using the sample plate of the embodiment.
[Explanation of symbols]
2 Capillary Array 10 Optical Measuring Unit 100 Sample Plate 101 Base Plate 102 Well 103 Well Guide 104 Electrode Plate 105 Recess 106 Plug 107 Hole for High Voltage Line Connection for Migration

Claims (2)

A disposable insulating resin base plate in which one or more holes are formed as wells, the bottoms of the wells are open, and each protrudes to the same height;
Indentations having dimensions smaller than the outer dimensions of the bottoms of the wells are formed at positions corresponding to the two-dimensional array of wells formed by arranging the base plates, and the bottoms of the wells are press-fitted into these indentations. And a conductive metal electrode plate for fixing the base plate. A plurality of capillary columns are arranged, and a plurality of samples are injected into the capillary columns one by one, and a multi-capillary array electrophoresis unit is simultaneously electrophoresed on all the capillary columns, and the capillary is irradiated with light in the multi-capillary array electrophoresis unit. In the multi-capillary electrophoresis apparatus having an optical measurement unit for measuring the absorbance of the irradiated part and the fluorescence from the sample,
The two-dimensionally arranged capillary column end is fixed downward on the sample injection side of the multi-capillary array electrophoresis section, and below the capillary column end there is a well containing a sample corresponding to the arrangement of the capillary column end. A sample plate arranged two-dimensionally, and a migration reservoir in which a buffer solution for electrophoresis is accommodated and voltage is applied to all capillary columns are arranged,
The sample plate is formed by arranging one or a plurality of holes as wells, a base plate made of a disposable insulating resin in which the bottoms of the wells are open and projecting to the same height, and the base plate. Indentations having dimensions smaller than the outer dimensions of the bottom of the well are formed at positions corresponding to the two-dimensional array of wells, and the bottom of the well is press-fitted into the depressions, thereby fixing the base plate. A metal electrode plate,
A multi-capillary electrophoresis apparatus comprising a moving mechanism that switches between the sample plate and the electrophoresis reservoir and contacts the end of the capillary column.

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