JPH07113786A - Electrophoresis device - Google Patents

Abstract

PURPOSE:To secure a large number of electrophoresis furnaces, and load simply a sample to be separated by connecting one ends of capillaries respectively to respective electrophorestic passages, enlarging sufficiently an interval between mutual other ends of the capillaries, and loading the sample through the capillaries. CONSTITUTION:An electrophorestic part 1 of a sample is plate gel constituted by sandwiching polyacrylamide between glass plates, and to one ends, capillaries 2 in which polyacrylamide is filled are connected, for example, by 100 pieces in a row in the vertical direction to the electrophoretic direction. The other ends of the capillaries 2 enter a sample tube 4 through a position determined by a support part 3. When sample liquid 5 is loaded, in a condition where the sample liquid is housed in the sample tube 4, prescribed voltage is impressed between electrodes 6 and 16 and between 16 and 12, and the sample liquid 5 is loaded. Afterwards, prescribed voltage is impressed between the electrodes 6 and 16 and between 16 and 12, electrophoresis is carried out, and the sample liquid 5 is electrophoresed in the capillaries 2 and the electrophorestic part 1, and molecular weight is separated.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoretic separation device for separating DNA or protein by electrophoresis.

[0002]

2. Description of the Related Art Conventionally, flat gel electrophoresis has been used for analyzing DNA and the like. In these methods, when loading the sample on one end of the gel, the sample was taken out from the sample tube with a pipette, and the sample was injected into the buffer solution in the well formed at the edge of the slab gel. Normally, a 0.3 mm thick polyacrylamide gel is used for DNA sequencing, but it is desired to apply a high electric field (200 V / cm) to the gel in order to increase the migration speed. When a high electric field is applied to a commonly used 0.3 mm thick gel, the gel is distorted due to heat generation and bubbles are generated in the gel, which does not work. Therefore, a thin-film gel (0.05-0.1) that generates less heat
(mm thickness) has been attracting attention.

Capillary gel electrophoresis, in which a capillary (inner diameter 0.05 to 0.1 mm) is filled with a gel, has been developed as a technique capable of high-speed electrophoresis. The capillary array method in which a large number of capillaries are arranged has attracted attention as a means for securing many migration paths in a narrow space. [Analytical Chemistry 64, 967 (1992) (Analytical Chemistry 64, 967)
(1992))].

[0004]

The capillary array method is the most effective method for achieving high speed and high throughput, but at present, it has good reproducibility and good quality (no bubbles, etc.) in a large number of capillaries. There is a problem that it is difficult to make the gel. On the other hand, the thin film gel has an advantage that a relatively good quality gel can be produced with good reproducibility. The loading by electrophoresis on a slab gel is currently performed manually using a pipette, but when the thickness of the gel is made thin (0.1 mm or less), it becomes difficult to load the sample to the gel edge. Come on. Therefore, how to easily load a sample on a thin flat gel of 0.1 mm or less becomes a problem.

In addition, regardless of the capillary gel electrophoresis or the slab gel electrophoresis, the electrophoretic apparatus is generally required to improve the throughput. One way to do this is to increase the number of lanes to be electrophoresed, so that many samples can be electrophoresed at one time.

However, if the number of lanes is simply increased, for example, 40 lanes with a width of 5 mm per lane will be used.
If you increase the number of lanes from 80 to 80, the required space is 2
From 00 mm to 400 mm, the area to be detected becomes wider. When using a detection system with the width of the light receiving element of 20 mm and imaging with a lens with an F value of 0.85, the reduction ratio m
Is 10 to 20, so the light collection efficiency when receiving light is 1 /
It is calculated by (4 * F * (1 + m)) ² , and it becomes about 1 / 3.6 from about 0.0715% to about 0.0196%, and the detection sensitivity decreases. Further, if the number of lanes is large, loading of the sample is troublesome, which is a problem.

Therefore, although 3 to 4 mm is currently required for each lane and the limit is about 40 lanes, the width and interval of the migration path can be made as small as possible while maintaining the size of the measurement area as it is. The major issue is how to secure the migration path. In addition, loading many samples at the same time
How to make it simple is also a big issue.

An object of the present invention is to provide an electrophoresis apparatus which can secure many migration paths and can easily load a sample to be separated by electrophoresis.

[0009]

[Means for Solving the Problems] In order to solve the above problems, many migration paths are densely secured in the electrophoretic portion, one end of each capillary is connected to each migration path, and the distance between the other ends of the capillary is set to be small. The electrophoretic device is configured so that it is sufficiently large and loading is performed through the thin tube.

[0010]

[Function] The thin tube connected to each migration path has an outer diameter of 0.35 mm.
Since it is thin enough, many migration paths can be secured in a fixed space. Also, since the thin tube can be flexibly bent,
Even if the migration path on the detection side is narrowed, the distance on the opposite side of the capillary, that is, the side on which the sample is loaded can be widened, and the sample can be placed on the edge of the gel as easily as the capillary array method. Can be loaded.

[0011]

【Example】

(Embodiment 1) A first embodiment according to the present invention will be described. 1 and 2 are explanatory views of the first embodiment. Sample migration part 1
Is a flat gel (thickness: 0.1 mm) composed of polyacrylamide sandwiched between glass plates. At one end, a thin tube 2 filled with polyacrylamide gel (inner diameter: 0.1 mm, outer diameter: 0.35 mm, long) 100 cm) are connected in a line perpendicular to the migration direction at intervals of 1.0 mm. In this way, the width of the migration lane is as narrow as about 10 cm as a whole, and when detecting a signal such as fluorescence from a sample during migration, a solid angle that can be detected can be easily made large. At the same time, since there are 100 migration lanes, which is far more than the conventional lanes of about 30, the throughput per hour is increased.

Here, the thin tube may be filled with a buffer solution instead of being filled with the gel, but the thin tube 2 is subjected to a surface treatment.
It is necessary to adjust the moving direction of the sample 5 toward the electrophoretic unit 1. In addition, the electrophoretic unit 1 may use a gel sandwiched between plastic sheets as a housing instead of the flat gel sandwiched by the glass plates.

The distance over which the sample migrates, that is, the distance from the end of the migration unit 1 to the detection unit 14 is 20 cm. FIG. 2A is a sectional view of the vicinity of the intermediate holder 10. The migration part 1 and the thin tube 2 are arranged face to face, and the joint part is occupied by the gel 7. The buffer solution 18 is on the gel 7, and the electrode 16 is in contact with the buffer solution 18. All of these are covered with the intermediate holder 10.

Without the electrode 16, when the voltage is applied between the electrode 6 and the electrode 12, the electric field strength in the thin tube 2 is larger than that in the electrophoretic section 1 because the resistance of the thin tube 2 is larger than that in the electrophoretic section 1. It may be larger, and the migration speed in the migration unit 1 may be slower than necessary. Alternatively, an excessive voltage must be applied between electrodes 6 and 12 to obtain the required migration rate. The presence of the electrode 16 allows the electric field strength in the electrophoretic section 1 and the electric field strength in the narrow tube 2 to be adjusted independently, so that the above problem does not occur.

Here, the distance between the electrophoretic section 1 and the thin tube 2 is 1 mm.
Below. It is desirable that this interval be narrow so that the sample migrates without diffusion. In this example, the gel 7 of the migration unit 1, the gel 7 in the intermediate holder 10 and the gel 7 in the thin tube 2 are used.
Is a continuous one polymerized at the same time. However, the gels that were originally polymerized separately may be contacted. For example, if the thin tube is filled with a thin gel and the flat plate is filled with a high gel, it is possible to quickly move the thin tube to the sample and separate the molecules in the flat plate.

The other end 11 of the thin tube 2 passes through the position determined by the supporting portion 3 and enters one each into a total of 100 sample tubes 4 as shown in FIG. 2 (b). . In this example, the arrangement of the holes of the supporting portion 3 through which the thin tube 2 passes is two-dimensional with 10 rows and 10 columns. The arrangement of the arrays is limited by the length of the thin tube, but may be one-dimensional broken lines or curved lines. It is also conceivable that the five divided holes are placed separately so as to surround the migration path, and at this time, the required length of the thin tube is shorter than that of this example.
The distance between the holes of the supporting portion 3 through which the thin tube 2 passes is 9 mm.

In the sample tube 4, the sample solution (or buffer solution) 5 and the end 11 of the capillary tube 2 are in contact with each other. An electrode 6 for applying a migration voltage is in contact with the sample liquid 5. In addition, the lower part of the electrophoretic part 1 of the flat gel is filled with the buffer solution 13
The electrode 12 is also immersed in the buffer solution 13. The three electrodes are energized by the power supply 15. FIG. 1C is a sectional view of the inside of the sample tube 4. The sample solution 5 is contained in the sample tube 4, and the electrode 6 and the thin tube 2 are immersed in the sample solution 5.

When the sample solution 5 is loaded, with the sample solution 5 to be loaded in the sample tube 4, a voltage of 3 kV is applied between the electrodes 6 and 16 and a voltage of 0 kV is applied between the electrodes 16 and 12. The voltage of 1 is simultaneously applied by the power supply 15 for 5 seconds to load the sample 2. When a sufficient amount of the sample solution 5 has entered the thin tube 2, the sample tube 4 is replaced with a sample tube containing a buffer solution.

Thereafter, a voltage of 8 kV is applied between the electrode 6 and the electrode 16 and a voltage of 2 kV is applied between the electrode 16 and the electrode 12 from the power source 15 at the same time to perform electrophoresis, and the capillary tube 2 and the migration section 1 are electrophoresed. The sample 5 is electrophoresed inside and the molecular weight is separated. Here, since the thin tube 2 is filled with gel, the place where the sample 5 is separated depending on the molecular weight is the migration unit 1.
Both inside and inside the thin tube 2 filled with the gel 7, there is no particular problem. Sample 5 electrophoresed while separating capillary 2
Migrates into the migration unit 1 immediately after leaving the capillary tube 2. When the thin tube 2 is filled with the buffer, the molecular weight is separated from the moment the sample 5 enters the migration unit 1.

(Embodiment 2) FIG. 3 is an explanatory view of the second embodiment. FIG. 3A shows a case where the capillary 2 is attached to the migration unit 1 in the direction perpendicular to the migration unit 1 in connection with the capillary 2 in FIG. FIG. 3B is a sectional view of the vicinity of the intermediate holder 10 'in the second embodiment. The migration path 1'and the intermediate holder 10 'of FIG.
Each hole is open. The holes are arranged along a flat plate at a 1 mm interval as in Example 1 and at right angles to the migration direction. The intermediate holder 10 'fixes the thin tube on a flat plate,
The gel (or buffer) 7 in the thin tube, the gel 7 in the flat plate, the buffer 18 ', and the electrode 16' are electrically contacted.

The roles of the buffer 18 'and the electrode 16' are the same as those of the buffer 18 and the electrode 16 of the first embodiment, respectively. Other parts are the same as those in FIG.

(Embodiment 3) In the third embodiment, an apparatus in which the thin tube 2 is removable in the first embodiment is constructed. The operation of this device is the same as that in the first embodiment until the sample solution 5 is loaded into the thin tube 2, but thereafter, the tip 11 of the thin tube 2 into which the sample is injected and the intermediate holder 1 are inserted.
Swap the other end of 0. Then, a voltage of 8 kV is applied between the electrodes 6 and 16, and a voltage of 2 kV is applied between the electrodes 16 and 12.
The above voltages are simultaneously applied from the power supply 15. Then, the sample liquid 5 contained in the thin tube 2 exits the thin tube 2 according to the electric field and enters the electrophoretic section 1. After this, the sample 5 is the migration unit
Separated by molecular weight in. In this embodiment, the thin tube 2
The required length of the thin tube 2 is shorter than that of the first embodiment.
Even if the gel 7 inside is broken, there is an advantage that the same thin tube 2 can be used many times by cutting that portion. As another configuration of this example, a device in which the thin tube 2 is removable in the second embodiment may be used.

[0023]

EFFECT OF THE INVENTION Samples can be easily loaded even on plate gels having an extremely thin thickness and plate gels having narrow migration lane intervals. It is possible to reduce the distance between lanes. This can increase the detection sensitivity in the fluorescence detection type electrophoretic device. Further, the number of migration lanes can be increased with respect to the same full-width migration space as in the past. Therefore, the throughput of the entire device is increased. In the present invention, the throughput is about 4 to 8 times because it is 0.5 to 1.0 mm / lane, which was about 4 mm / lane in the past.

[Brief description of drawings]

FIG. 1 is an explanatory diagram of the vicinity of an intermediate holder according to a first embodiment of the present invention.

FIG. 2 is a sectional view of the vicinity of a sample tube of Example 1.

FIG. 3 is an explanatory diagram of the vicinity of an intermediate holder according to a second embodiment of the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Electrophoresis part, 2 ... Gel filling thin tube, 3 ... Capillary support part, 4 ... Sample tube, 5 ... Sample liquid or buffer liquid, 6 ... Electrode, 10 ... Intermediate holder, 11 ... Tip of the thin tube,
12 ... Electrode, 13 ... Buffer solution, 14 ... Detection part, 15 ...
Power supply.

Claims (5)

[Claims] 1. An electrophoretic device comprising: an electrophoretic unit for separating a sample; and a sample guide unit composed of a thin tube provided for guiding the sample to the electrophoretic unit. 2. The sample guide portion according to claim 1,
Electrophoresis device consisting of gel-filled capillaries. 3. The sample guide section according to claim 1,
Electrophoresis device consisting of a buffer-filled capillary. 4. The electrophoretic device according to claim 2, wherein the electrophoretic section for separating the sample comprises an electrophoretic path sandwiched by an insulator such as a glass plate or a plastic plate. 5. The electrophoresis device according to claim 4, wherein the thin tube of the sample guide portion is removable.