JPH0593711A - Narrow-groove type electrophoresis device - Google Patents

Abstract

PURPOSE:To obtain a narrow-groove type electrophoresis device having high sensitivity and throughput. CONSTITUTION:Numerous capillary electrophoresis paths are formed of numerous narrow grooves 3 by putting a glass plate 1 with a laser irradiation groove 2 and the numerous narrow grooves 3 upon a plate having no groove. Since the cross section of the electrophoresis path of this narrow-groove type electrophoresis device can be reduced, the volume of a DNA band can be reduced and a small amount of sample can be measured. In addition, the throughput of the electrophoresis device can be improved.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for separating and detecting a fluorescently labeled biological sample such as DNA by electrophoresis, for example, a DNA sequencer or a gene diagnostic apparatus.

[0002]

2. Description of the Related Art Conventionally, the base sequence of DNA has been determined by labeling the DNA with a radioactive element, performing gel electrophoresis separation, and then transferring it to a separation pattern film. However, in addition to the complexity of using a radioactive label, there is a problem that it takes time and labor, and a method using real-time photodetection using a fluorescent label is beginning to be used.

[0003]

The method using such a fluorescent label and a flat plate gel (slab gel) has a drawback in that high sensitivity cannot be obtained. Therefore, a technique for reducing the measurement volume and increasing the sensitivity is to use capillary electrophoresis. Has been done. However, in the method using capillary electrophoresis, the migration path is set to 1
Since only books can be secured, throughput is small and D
In NA sequencing, it is necessary to use fluorophores having different emission wavelengths to identify the terminal base species A, C, G, and T, and it is difficult to realize all combinations of light sources and fluorophores with good excitation efficiency. is there.

An object of the present invention is to solve this problem and provide a separation and detection apparatus for DNA and the like with high sensitivity and high throughput.

[0005]

The above object can be achieved by forming a large number of grooves on a quartz or glass plate and forming a gel in the grooves to form a capillary-type migration path.

[0006]

[Function] Many straight lines (or curves) dug on a quartz plate
Each groove has the same role as the capillary migration path. Since at least 10 or more grooves can be dug per 1 cm, 100 or more migration paths can be secured in a region with a width of 10 cm. By reducing the diameter of the groove, the volume of the DNA band can be reduced. The detection limit is determined by the background light and the magnitude of fluorescence from the target. Therefore, a small amount of detection can be realized by pushing the sample into a small volume.

[0007]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to FIGS. On one side of the glass plate 1 which is the gel holding plate shown in FIG. 1, grooves 3 having a width of 0.2 mm and a depth of 0.2 mm are formed at intervals of 1 mm to form a glass plate with fine grooves. The spacing can be 0.5 mm or less. A groove 2 (width 0.4 mm, depth 0.3 mm) for laser irradiation is provided 25 cm from the upper end at a right angle to the migration path. The upper end is the sample injection part, and the end 30 of each groove 3 is a skirt. It is spread out in a shape. The end face has a cross section of about 0.5φ so that sample injection can be performed easily. As shown in FIG. 2, this narrow grooved glass plate 1 is closely adhered to a flat plate 4 of glass having no groove to form a capillary migration path. A polyacrylamide gel is formed in these migration paths and set as shown in FIG. An electrophoretic plate composed of a laminated body of glass plates 3 and 4 is erected vertically, and an upper buffer tank 13 is placed so that the buffer solution is in contact with the upper end of each electrophoretic path on which a gel is formed.
Is provided. The lower end of the migration path is at the bottom buffer tank 1.
It is installed so as to be in contact with the buffer solution of 2. A sample such as DNA is added to the upper end of the gel, and an electrophoretic voltage is applied between the buffer tanks 13 and 12 to perform electrophoretic separation. In the examples, DNA labeled with TexaoRed (excitation maximum wavelength 596 nm, emission maximum wavelength 615 nm) was electrophoresed and separated from the top. The PMS (Particle Measurement) is used as the excitation light source 5.
Systems) He-Ne laser (oscillation wavelength 594n
m, output 2.5 mW). The laser light is reflected by the mirror 6 and made incident on the side surface of the migration plate so as to pass through the groove 2 so that all migration paths are uniformly irradiated. The fluorescence-labeled DNA fragment emits fluorescence when passing through the laser irradiation portion, but the fluorescent image from the linear irradiation portion, that is, the groove 2 passes through the wavelength selection filter 8 and is then formed on the light receiving surface of the image amplifier 11 by the lens 9. It is imaged and detected by a high sensitivity line sensor, eg consisting of a diode array, or a two-dimensional photodetector 10, eg a CCD. FIG. 3 shows the intensity distribution of the fluorescence image during one period during the measurement, and fluorescence can be seen from the migration path portion during the passage of the fluorescence-labeled DNA fragment.
FIG. 4 shows an example in which λ phage was cleaved with a restriction oxygen HindIII as a sample, and a DNA fragment having a fluorescent label at the cleaved portion was electrophoresed. It shows the time change of fluorescence emitted from a specific migration path and corresponds to a DNA fragment spectrum. DNA
The width of the band is about 0.7mm and 2x10 per band
^It contains only a small amount of ^-19 mole DNA, but the peak is well detected in S / N.

Although the electrophoretic plate is used vertically in the above embodiment, it is also possible to operate the electrophoretic plate as shown in FIG. 5 in a horizontal position. In this electrophoretic plate, another glass plate 4'which is closely attached to the glass plate 1 with fine grooves having grooves 3 is provided with a hole 1 to be a sample injection port as shown in FIG. 5 (a).
6 is provided corresponding to each groove 3, and there is an advantage that a sample can be easily injected.

Further, at the position corresponding to the position close to the other end of each groove 3, a rectangular hole 15 for allowing the migration of the electrophoretic fragment is provided in common to each groove. The narrow grooved glass plate 1 and the glass plate 4'are brought into close contact with each other, and a gel is formed in the capillaries generated thereby and the holes 15 and 16 to form a plurality of capillary migration paths. The groove 3 may be provided from the position corresponding to the sample injection port 16 to the position corresponding to the fragment outlet port 15. However, as shown in FIG.
May be provided from one end to the other end. In any case, the migration path extends from the sample injection port to the fragment outlet, and the sample injection port 16 is not the end surface of the glass plate 4 but the glass plate 4
Since they are lined up on the main surface of ′, sample injection becomes easy. Such an electrophoretic plate is installed horizontally as shown in FIG. 5B, and buffer tanks 13 ′ and 12 ′ are installed at the position of the sample injection port 16 and the position of the fragment outlet 15. After the sample solution to be analyzed is injected into the sample injection port 16, each buffer tank is filled with the buffer solution, and electrophoresis is performed by applying a voltage to irradiate the groove 2 with laser light to detect passage of fragments. In these examples DN
A is labeled with a multicolor fluorescent light, and the emitted fluorescence is wavelength-selected and received using a prism or the like to improve the throughput or A, C, G, T, D in DNA sequencing.
It is also possible to provide a function of identifying the NA terminal by the difference in emission wavelength.

Although polyacrylamide gel is used as the electrophoretic medium in the above embodiments, it is obvious that a gel such as agarose or a conductive liquid may be used.

In the above embodiments, the grooves are formed in the gel holding plate (flat plate), but the flat plate with fine grooves has a linear or ribbon-shaped protrusion fixed to the flat plate as in the embodiment shown in FIG. Alternatively, glass wires, polymer wires or ribbons 17 (made of polyethylene terephthalate, acrylic, etc.) or other wires or ribbons made of an insulating material or a high electric resistance material are sandwiched and held between flat plates, and a large number of migration paths 3 are provided.
Can also be formed. Ribbons and wires serve to separate each migration path. In the embodiment shown in FIG. 6, the flat plate with the narrow groove and the flat plate are closely adhered to each other to form a capillary migration path. Even if they are overlapped with each other, the capillary migration path can be similarly formed.

A glass material such as quartz glass or heat-resistant tempered glass is suitable for the flat plate 3 having the groove and the flat plate 4 or 4'which is closely adhered to the flat plate 3, but any insulating material having a certain degree of thermal conductivity may be used. Can be used if If an etchable photosensitive glass is used, a large number of grooves can be accurately formed by etching with good reproducibility. On the other hand, even a plate material having conductivity can be used by coating the surface of the plate or the plate with grooves and then coating the surface with an insulating material. For example, when a metal plate whose surface is an oxide film or a nitride film as an insulating layer is used for any of the flat plates, it is advantageous for discharging heat generated by electrophoresis.

In addition to the side incidence method shown in FIG. 1, an excitation light scanning method may be used for detecting DNA and the like.

[0014]

According to the present invention, the cross-sectional area of the migration path can be restricted to a small value by the groove, and the volume of the DNA band can be reduced, so that it is possible to measure with a small amount of sample.
For example, in the conventional DNA measurement using a flat-plate gel, a gel thickness of 0.3 to 0.5 mm and a DNA band width of 2 to 5 mm were used. That is, the cross-sectional area of the migration path was within 0.6 mm ² even if it was small. On the other hand, in the present invention, in the embodiment, it is 0.04 mm ² , and by further reducing the width of the groove, 0.01 mm ²
Alternatively, it is possible to obtain a migration path cross-sectional area smaller than that. By making the area small, it became possible to measure with a trace sample of 1 to 2 digits. Further, since 1 to 2 grooves can be formed per 1 mm, the number of migration paths can be increased to 100 or more, which was conventionally able to secure only 20 to 30 per 10 cm, which is very effective in improving the throughput. In addition, since the migration path interval is 1 mm or less, the temperature difference is small, and since there is no difference in the migration speed of DNA depending on the migration path, the DNA is labeled with one type of fluorophore and the terminal DNA base species is identified by the difference in the migration path. It can also be used effectively for sequencing.

[Brief description of drawings]

FIG. 1 is a plan view of a grooved glass plate.

FIG. 2 is a conceptual diagram of a measuring device.

FIG. 3 shows an example of a measured signal.

[Fig. 4] DN in which change in fluorescence intensity at a specific position is measured
A fragment spectrum.

FIG. 5 shows an example of a horizontal migration tank.

FIG. 6 is a sketch of a migration plate with migration path separation ribbon.

[Explanation of Codes] 1 ... Glass plate with fine groove, 2 ... Laser irradiation groove, 3 ... Migration fine groove, 4 ... Flat plate, 5 ... Laser, 6 ... Mirror, 7 ... Laser light, 8 ... Filter, 9 ... Lens 10 ... One-dimensional or two-dimensional photodetector, 11 ... Image amplifier, 12 ... Lower buffer tank, 13 ... Upper buffer tank, 14 ... DN
A fragment peak, 15 ... DNA fragment outlet, 16 ... DNA
Fragment injection port, 17 ... Electrophoresis separation ribbon.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location 7235-2J 331 Z

Claims (8)

[Claims] 1. The sample fragment is detected by irradiating a detection position in the middle of a migration path along which a sample fragment migrates with laser light and detecting a real-time transition of the emission of a fluorescent labeling substance caused by the irradiation of the laser light. In an electrophoretic device for real-time detection that obtains information that a position has been passed, the electrophoretic device has a first flat plate having fine grooves on its surface, and a second flat plate in close contact with the first flat plate. A narrow groove type electrophoretic device characterized in that a fine tube formed of the first and second flat plates is used as a migration path. 2. The narrow groove type electrophoretic device according to claim 1, wherein the migration path is filled with gel. 3. The first flat plate is provided with a plurality of grooves serving as migration paths, and a groove for passing the laser light in a direction perpendicular to the plurality of grooves. The narrow groove type electrophoretic device according to. 4. The narrow groove type electrophoretic device according to claim 1, wherein the second flat plate is provided with a sample injection port and a fragment outflow port corresponding to the positions of the grooves. 5. The narrow groove type electrophoretic device according to claim 1, wherein at least one of the first and second flat plates is made of a glass material. 6. The narrow groove type electrophoretic device according to claim 1, wherein at least one of the first and second flat plates is a metal plate whose surface is coated with an insulating material. 7. The narrow groove type electrophoretic device according to claim 1, wherein the groove of the first flat plate is formed by cutting the flat plate. 8. The groove of the first flat plate is formed by fixing a linear or ribbon-shaped projection to the flat plate, or a ribbon or a wire-shaped product is sandwiched between two flat plates to form a migration path. The narrow groove type electrophoretic device according to claim 1, wherein