JPH10132785A - Electrophoresis device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make the sample loading work simple and efficient, by making a migration board holding a gel electrolyte layer a hollow rectangular columnar body whose both ends in the longitudinal direction are opened. SOLUTION: A migration board 1 in the shape of a strip has openings at both ends 3, 3 in its longitudinal direction and is a hollow columnar body having a hollow part, and the inside of the hollow part is filled up with a gel electrolyte such polyacrylic amide gel and so on. It is possible to perform filling up merely by a capillary phenomenon, and a gel electrolytic solution does not leak out because of the caselike shape. When a sample is loaded on the migration board 1, extremely thin pipes (outside diameter 0.2mm) of capillaries 7 for example are used, and the capillaries are arranged at spaces of 0.3mm. Besides, for one sample, a migration path is provided to each of the four bases of adenine, thymine, cytosine, and guanie, and at either end a migration path is provided for smiling correction. Consequently, it becomes possible to analyze 10-14 pieces of samples simultaneously, when the breadth of the hollow part is 18mm. Accordingly, the sample loading work becomes simple and efficient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an electrophoresis apparatus. More specifically, the present invention relates to a gel electrophoresis apparatus using an improved electrophoresis plate.

[0002]

2. Description of the Related Art Gel electrophoresis is widely used as a method for determining a base sequence of DNA or the like.

Conventionally, when performing electrophoresis, a sample is labeled with a radioisotope and analyzed, but this method has a drawback that it requires time and effort. In addition, maximum safety and control are always required from the viewpoint of radioactivity management, and analysis cannot be performed unless in a special facility. For this reason,
Recently, a method of labeling a sample with a phosphor has been studied.

In the method using light, a fluorescently labeled DN is used.
The A fragment is allowed to migrate in the gel.
A photoexciting section and a photodetector are provided for each electrophoresis path below ２０20 cm, and DNA fragments passing therethrough are sequentially measured. For example, DNAs of various lengths whose terminal base species are known are duplicated by an enzymatic reaction (dideoxy method) using a DNA chain whose sequence is to be determined as a template, and these are labeled with a fluorescent substance. That is, adenine (A) labeled with a fluorescent substance
A fragment group, a cytosine (C) fragment group, a guanine (G) fragment group, and a thymine (T) fragment group are obtained. These fragment groups are mixed and injected into separate electrophoresis lane grooves of an electrophoresis gel,
Apply voltage. Since DNA is a chain polymer polymer having a negative charge, it moves in the gel at a speed inversely proportional to the molecular weight. Shorter (small molecular weight) DNA strands move faster and longer (larger molecular weight) DNA chains move more slowly, so that DNA can be fractionated by molecular weight.

Japanese Patent Application Laid-Open No. 63-21556 discloses that a line on a gel of an electrophoresis apparatus irradiated with a laser and an arrangement direction of a photodiode array are perpendicular to a migration direction of a DNA fragment in the electrophoresis apparatus. Is disclosed.

FIG. 7 is a schematic diagram for explaining the configuration of the apparatus. The electrophoresis plate 74 has an electrophoresis gel (for example, polyacrylamide) sandwiched between two glass plates. The thickness of the entire electrophoresis plate is about 10 mm, but the thickness of the gel electrolyte layer itself is less than about 1 mm. An upper end of the comb-like gel electrolyte layer exists slightly below the upper end of the electrophoresis plate 74. In the comb-shaped groove 75, a DN labeled with a fluorescent substance is used.
Inject fragment A. The comb-shaped groove 75 can be easily formed by inserting a tool such as a shark comb (not shown) into the surface of the gel electrolyte layer.

In the apparatus shown in FIG. 7, a laser beam emitted from a light source 70 is reflected by a mirror 72 and irradiates a predetermined point in the gel of the electrophoresis plate 74 horizontally and horizontally. When the fluorescently labeled DNA fragment 76 that has migrated through the gel passes through the irradiated area, fluorescence is sequentially emitted from the DNA fragment. At this time, the type of the terminal base can be determined from the horizontal position of the fluorescence emission, the length of the fragment can be determined from the difference in the migration time from the start of the migration, and the sample can be further identified by the emission wavelength.
The fluorescent light from each migration path forms an image at the light receiving section 82 of the image intensifier 80 by the lens 78. This signal is amplified, converted into an electric signal by the photodiode array 84, and measured. The sequence of each DNA fragment is calculated by computer processing the measurement results, and the target DN is calculated.
The nucleotide sequence of A is determined.

In the case of the electrophoresis plate shown in FIG. 7, it takes time and effort to construct the electrophoresis plate by combining two flat glass plates and a spacer for securing the gel thickness. If the gel electrolyte is to be automatically filled in the gap between the two flat glass plates, the gel electrolyte solution leaks from the gap between the glass plate and the spacer, so that it is difficult to automatically manufacture the electrophoresis plate.

In the case of a plate-like electrophoresis plate, the electrophoresis path gradually changes from a linear state due to factors such as temperature conditions, temperature distribution, and gel dissolution, gradually curves downward, and moves in one of the left and right directions. May shift to Such a shift of the migration path is generally called “smileing”. When smile occurs, the fluorescence generation position when the laser excitation light is incident on the DNA sample is shifted from the arrangement position of the fluorescence detection means, so that the fluorescence detection rate decreases, and as a result, the DNA base sequence determination accuracy is poor. become.

In order to solve such a problem of the flat electrophoresis plate, it has been attempted to use a plurality of capillaries (ultrafine tubes) corresponding to each electrophoresis path in a group. However, since a capillary (ultra-fine tube) having an inner diameter of φ100 μm or less is usually used, there are the following technical problems. When irradiating a fluorescence-labeled DNA sample with excitation light, it is difficult to uniformly irradiate a plurality of samples (capillaries) with excitation light. Therefore, it is difficult to increase the number of samples. It is very difficult as a matter of fact to match the electrophoretic conditions between the capillaries, so that it is not possible to establish a mutual relationship between electrophoretic data. Loading of sample is difficult due to small tube diameter.

[0011]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a novel electrophoretic means which can solve various problems of the conventional flat and capillary electrophoretic means. is there.

[0012]

The above object is achieved by an electrophoresis plate comprising a hollow rectangular tube having both ends opened in the longitudinal direction.

[0013]

FIG. 1 is a plan view of an electrophoresis plate according to the present invention, and FIG. 2 is a sectional view taken along line AA in FIG. The appearance of the electrophoresis plate 1 of the present invention is generally a strip shape.
Both ends 3, 3 in the longitudinal direction of the strip-shaped electrophoresis plate 1 are open. The electrophoresis plate 1 of the present invention has a hollow portion 5 as shown in FIG. Therefore, the electrophoresis plate 1 of the present invention is a hollow cylindrical body. The inside of the hollow portion 5 is filled with a gel electrolyte such as polyacrylamide gel. The length L of the electrophoresis plate 1 is not particularly limited. The length may be any length necessary and sufficient for separating the sample. For example, it can have a length in the range of 50 mm to 400 mm, preferably 100 to 300 mm. Width W ₁ of the electrophoresis plate 1 is not particularly limited,
Generally, it is preferable to be about 20 mm. Since the width of a conventional flat electrophoresis plate is generally 200 mm,
The electrophoresis plate 1 of the present invention is about 1/10 the size of the conventional electrophoresis plate. The thickness T ₁ of the electrophoresis plate 1 of the present invention is, for example, 2.1
mm. The thickness T ₂ of the hollow portion 5, for example, can be set to 0.1 mm. Of course, T ₁ and T ₂ other than these can also be used. T ₂ is at least 100 μm
Can be thinned. The width W ₂ of the hollow portion 5, for example, can be set to 18 mm. W ₁ and W ₂ other than the values can of course also be used.

The material for forming the electrophoresis plate 1 of the present invention is not particularly limited. For example, it can be formed from glass such as silica glass or quartz glass, or a transparent synthetic resin such as an acrylic resin or a polycarbonate resin. If molded with synthetic resin, the production cost is low, so the electrophoresis plate 1 of the present invention is used.
Can be disposable. As a result, gel treatment after use becomes unnecessary, and the throughput of the analysis operation is improved.

Since the electrophoresis plate 1 of the present invention is cylindrical, even if the gel electrolyte solution is filled in the hollow portion 5, the gel electrolyte solution never leaks unlike the conventional electrophoresis plate. The filling of the hollow portion 5 with the gel electrolyte solution can be carried out by simple capillary action, or by immersing one opening in a container containing the gel electrolyte solution and vacuum-suctioning the other opening.

When loading a sample onto the electrophoresis plate 1 of the present invention, for example, as shown in FIG. 3, an ultrafine tube such as a capillary 7 can be used. When the capillary 7 has an inner diameter of about 0.1 mm (outer diameter of 0.2 mm) and an interval between the capillaries of 0.3 mm, each electrophoresis path is set to each of four bases A, T, C, and G for one sample. Assuming that one migration path is provided at each end and used for correcting the smile, 0.3 × (10 samples × 4 migration paths + two migration paths at both ends) = 12.6 mm.
Therefore, when the width W ₂ of the hollow portion is 18 mm,. 10 to
Up to about 14 samples can be analyzed simultaneously. That is, when a sample is loaded by a capillary, almost the same number of samples can be analyzed with one strip-type electrophoresis plate of the present invention as with one conventional plate-type electrophoresis plate, so that the entire electrophoresis apparatus can be reduced in size. It becomes possible.
Moreover, sample loading onto the gel electrolyte layer only requires inserting the capillaries into the electrolyte layer, so that the sample loading operation on the electrophoresis plate of the present invention is very simple and easy as compared with the sample loading operation on the conventional flat electrophoresis plate. It is efficient.

The sample can be loaded using a conventional shark comb (not shown) without using the capillary 7. In this case, the minimum distance between the migration paths is 2.8 mm. Therefore, 2.8x
(1 sample x 4 migration paths + 2 migration paths at both ends) = 16.8 m
m. Therefore, when the width W ₂ of the hollow portion is 18 mm, when loading the sample using shark comb, samples every one must use one electrophoresis plate. That is, an electrophoresis plate is required for each sample.

When a sample is loaded using the capillary 7, the loading can be performed as shown in FIGS. Both ends of the electrophoresis plate 1 are inserted into the substantially L-shaped support sockets 9 and 11, respectively. The support sockets 9 and 11 are provided with through holes 13 communicating with both ends thereof. The electrophoresis plate 1 is inserted into the through hole 13 from one end of the support sockets 9 and 11. The other ends of the support sockets 9 and 11 are connected to the buffer solution 19 in the buffer tanks 15 and 17.
Immersed in. Buffer solution 19 is supplied to support sockets 9 and 1
1 rises from the other through hole 13 by capillary action, and contacts the end of the electrophoresis plate 1 in the through hole 13.

At the upper part of the support socket 9 in the buffer tank 15, a capillary insertion port 21 communicating with the through hole 13 is provided. The capillary 7 is inserted through the capillary insertion port 21 and is brought into contact with the gel electrolyte layer at the end of the electrophoresis plate 7 inserted into the through hole 13 of the support socket 9. The other end of the capillary 7 is immersed in the sample 25 filled in each hole of the microplate 23. At this time, the inside of the capillary 7 is filled with a buffer solution. When a voltage is applied between the microplate 23 and the buffer tank 15, the sample 25 is loaded on the gel electrolyte layer of the electrophoresis plate 1 by the capillary 7.

Next, the buffer tanks 15 and 17
When a voltage is applied between the sample and the sample, the sample migrates in the gel electrolyte layer of the electrophoresis plate 1 from the buffer tank 15 toward the buffer tank 17 and is separated.

As shown in FIG. 5, an excitation light source 27 and a fluorescence detecting means 29 are arranged at appropriate positions on the electrophoresis plate 1 so as to face vertically. As the excitation light source 27,
A gas laser such as He-Ne or a semiconductor laser can be used preferably because the fluorescent dye can be efficiently excited. In addition to the method of irradiating from the wide surface of the electrophoresis plate 1 (surface irradiation) as shown in FIG. 5, the excitation light is transmitted through the gel electrolyte layer 30 from the side surface of the electrophoresis plate 1 as shown in FIG. A scheme can also be used.

As the fluorescence detecting means, all known types of detecting means can be used. For example, JP-A-7
A fluorescence detecting means including a gradient index lens array, a filter and a CCD line sensor as described in JP-A-98276 is suitable. In this case, it is preferable to use the pixels of the CCD line sensor as 7 × 7 μm.

The electrophoresis plate 1 of the present invention can be used in an upright state, similarly to the electrophoresis plate 74 shown in FIG. In this case, a groove for sample injection can be formed at the upper end of the gel electrolyte layer of the electrophoresis plate 1 using a shark comb. Even when the electrophoresis plate 1 is used upright, the excitation light source and the fluorescence detection means can be arranged so as to face each other in the side direction or the front direction with the electrophoresis plate interposed therebetween.

[0024]

As described above, according to the present invention,
Extremely compact compared to conventional flat plate electrophoresis plates,
In addition, sample loading is simple and can be performed automatically or semi-automatically. In addition, since the electrophoresis plate itself is cylindrical, filling with the gel electrolyte solution is easy, and the production of the gel electrolyte layer is performed easily and in a short time. When the number of samples to be analyzed is increased, it is possible to cope with the problem simply by adding and arranging a cylindrical electrophoresis plate and a buffer tank, which is efficient. Since the rigidity is higher than that of a conventional flat electrophoresis plate, a thin-walled structure is possible. For this reason, it is easy to control the temperature of the entire electrophoresis plate.

[Brief description of the drawings]

FIG. 1 is a plan view of an example of an electrophoresis plate of the present invention.

FIG. 2 is a sectional view taken along the line AA in FIG.

FIG. 3 is a partial plan view showing a state where a sample is loaded on a migration plate of the present invention using a capillary.

FIG. 4 is a plan view showing an example of an apparatus configuration for loading a sample onto a migration plate of the present invention using a capillary and performing electrophoresis.

FIG. 5 is a sectional view taken along line BB in FIG. 4;

FIG. 6 is a cross-sectional view showing a state where an excitation light source and fluorescence detection means are arranged on both sides of the side surface of the electrophoresis plate.

FIG. 7 shows a D disclosed in Japanese Patent Application Laid-Open No. 63-21556.
FIG. 2 is a schematic configuration diagram of an NA base sequencer.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Electrophoresis plate of the present invention 3 Opening 5 Hollow 7 Capillary 9, 11 Support socket 13 Through hole 15, 17 Buffer tank 19 Buffer solution 21 Capillary insertion port 23 Microplate 25 Sample solution 27 Light source for excitation 29 Fluorescence detecting means

Claims (5)

[Claims] 1. An electrophoresis apparatus using a gel electrolyte layer for electrophoretically separating a sample, wherein the electrophoresis plate holding the gel electrolyte layer has a hollow rectangular shape having open ends at both longitudinal ends. An electrophoresis apparatus, comprising: 2. The electrophoresis apparatus according to claim 1, wherein the electrophoresis plate is made of glass. 3. The electrophoresis apparatus according to claim 2, wherein said electrophoresis plate is made of quartz glass. 4. The electrophoresis board according to claim 1, wherein said electrophoresis board is made of synthetic resin.
Electrophoresis device. 5. The electrophoresis apparatus according to claim 4, wherein the electrophoresis plate is made of an acrylic resin.