JPH08261988A - Stacked migration medium and light detecting electrophoretic device - Google Patents

Abstract

PURPOSE: To concurrently acquire much information from many migration plates with one measuring device by holding the migration plates formed with multiple migration paths between multiple holding plates arranged in parallel at prescribed intervals. CONSTITUTION: Multiple (e.g. four) gel plates (migration plates) 4 filled with an unpolymerized material of polyacryl amide gel, for example, are formed between holding frames (glass plates) arranged in parallel at intervals of about 0.3mm in a migration holding frame, and sample injection grooves are formed on the surfaces. The migration plates 4 are erected in a lower buffer tank 6, DNA fragments fluorescence-marked for determining the base sequence are injected into the upper sections of the migration plates 4 from the openings on the side faces of upper buffer tanks 5, a voltage is applied between electrodes, and the DNA fragments are migrated downward. When a laser 1 is radiated to the migration flow, the DNA fragments migrating in four different migration paths for four terminal base species generate fluorescence, images are formed on a two-dimensional detector 8, and four fluorescent images 13 are displayed 12 and processed 10 at the same time.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a photodetection electrophoretic device and electrophoretic medium, and more particularly to a photodetection electrophoretic device and electrophoretic medium suitable for separating and detecting fluorescently labeled DNA, RNA or protein. is there.

[0002]

2. Description of the Related Art Conventionally, autoradiography has been used to determine the base sequence of DNA. Recently, simpler optical detection methods using fluorescent labels have begun to be used (Natu
re 321: 674-679 (1986), Bio / Technology 6,816,198
8). In this method, a fluorescently labeled DNA fragment is electrophoresed in a plate-type gel separation plate of an electrophoretic plate of about 20 cm × 40 cm, and a downstream of a certain distance of about 20 cm from the starting point of electrophoresis is irradiated with a laser, and the migration direction of the DNA fragment is determined. The direction of the DNA fragment is detected in a direction substantially perpendicular to the direction, that is, the detector provided on the front surface of the gel plate receives the fluorescence from the fluorescently labeled DNA fragment passing therethrough, and the length of the DNA fragment is known from the migration time of the DNA fragment. It determines the base sequence.

[0003]

In the apparatus using the above-mentioned conventional technique, one electrophoretic plate needs to be provided with one measuring section, and information from many electrophoretic plates cannot be obtained by one measuring section. There wasn't. For this reason, there has been a problem that many devices are required to measure many samples. Also,
In the above technique, the number of migration paths that can be secured on one migration plate is limited, and it is difficult to measure many samples at the same time. The object of the present invention is to solve these problems of the prior art,
It is an object of the present invention to provide a photodetection electrophoretic device and an electrophoretic medium that can obtain information from a large number of electrophoretic plates at the same time with one measuring device and that is convenient for measuring a large number of samples.

[0004]

In order to achieve the above object, a laminated electrophoretic medium of the present invention has a plurality of holding plates laminated in parallel with a predetermined gap, and a plurality of holding plates held in the gap. A plate-shaped migration member is provided, and a plurality of migration paths are set in each of the plate-shaped migration members.

Further, the photodetection electrophoretic device of the present invention comprises an electrophoretic separation unit for electrophoretically separating a fluorescently labeled sample, means for applying a voltage to the electrophoretic separation unit, and photoexcitation of the sample separated by the electrophoretic separation unit. In the photodetection electrophoretic device including the means for generating fluorescence and the means for detecting fluorescence, the electrophoretic separation unit is provided with the above-mentioned laminated electrophoretic medium.

The fluorescence detector is installed at a position where the fluorescence emitted from the end faces of the plurality of migration paths in the migration direction or the vicinity of the end faces are individually and simultaneously received and detected. As a preferred embodiment regarding the installation position of the fluorescence detector, the light-receiving unit of the fluorescence detector is configured to be installed at a position facing the end face in the migration direction of a plurality of gel plates arranged in parallel, more specifically, As shown in FIG. 1, the lower part of a plurality of gel plates arranged side by side is located in a transparent lower buffer tank, and the light receiving part of the fluorescence detector is installed to face the bottom surface of the lower buffer tank. Or
Reflecting each fluorescence emitted from the end surface or the vicinity of the end surface in the migration direction in each of the plurality of gel plates arranged in parallel,
A configuration in which a mirror that allows light to enter the light receiving portion of the fluorescence detector is provided can be given.

As the fluorescence detector, a one-dimensional or two-dimensional fluorescence detector can be used. Further, in the photodetection electrophoretic device of the present invention, it is provided with a means for irradiating at least one end of the plurality of gel plates arranged in parallel in the migration direction or the vicinity of the end at the same time in at least the same gel plate plane, and the fluorescence detector. It is desirable to have an optical system that simultaneously receives a plurality of fluorescent ray images.

As another preferred embodiment of the photodetection electrophoretic device of the present invention, as shown in FIGS. 4 to 6, a plurality of gel plates are integrally laminated with each other via gel holding plates. Can be configured. The gel holding plate can be composed of a glass plate, a quartz plate, or the like. It should be noted that 20 to 30 sample injection wells are formed on the gel plate in the photodetection electrophoresis apparatus of the present invention, and the fluorescence-labeled sample is injected into this well. Further, as the gel, a 4 to 8% polyacrylamide gel or 0.
A 3 to 1% agarose gel is preferably used.

As a sample to be separated and detected by the photodetection electrophoresis apparatus of the present invention, DNA whose base sequence is to be determined
Alternatively, RNA can be used, but proteins and the like can also be used as the target sample. Further, when the sample for fluorescence detection is a multi-color labeled sample, each fluorescence emitted from the end face in the migration direction of each of the plurality of gel plates arranged in parallel is the imaging site of the fluorescence detector. Each label can be identified and detected by interposing a means for dispersing the respective fluorescences according to wavelength in the passage leading to.

The laminated electrophoretic medium of the present invention is shown in FIGS.
It can be constructed practically by the holding container shown in. This holding container, as shown in FIGS. 4 and 5,
A plurality of gel holding plates are erected at a predetermined interval on the bottom surface of the holding container, and the upper part of the gel holding plate erected in the holding container has a space capable of holding an electrolytic solution. An injection port for the unpolymerized gel material is provided in the lower part, and a communication for allowing the unpolymerized gel material injected from the injection port to flow between the plurality of gel holding plates at the bottom of the holding container. Has a groove.

When a plurality of gel plates are arranged side by side and a certain distance from the starting point of migration is irradiated with light, fluorescence is emitted from that part. When viewed from the direction perpendicular to the surface of the gel plate, these fluorescent line images overlap with each other, so that individual information cannot be obtained. However, when viewed from the end surface side in the migration direction of each gel plate, these can be observed separately. In the photodetection electrophoretic device of the present invention, the high-sensitivity two-dimensional sensor is located at a position where the fluorescence emitted from the end surface or the vicinity of the end surface is individually and simultaneously received and detected, that is, for example, a position facing the end surface. Since such detectors are installed, it is possible to simultaneously detect fluorescent images from different migration plates in the vertical direction as a line image by one detector.

Further, when the number of the plurality of gel plates is n, the same number of migration paths as when the width of the gel plate is n times can be ensured, so that a large number of samples can be measured. Also, when detecting a wide gel plate with a line sensor, it is necessary to use a long line sensor or use a lens with a large image reduction rate to convert the fluorescent image into a small image. Although there is a technical problem, in the present invention, since a large number of short fluorescent line images are detected, there is no need to increase the image reduction rate of the lens, which is convenient for obtaining high sensitivity.

In fluorescence detection, it is very important to obtain high sensitivity. It is necessary to increase the amount of received light to achieve high sensitivity. The amount of received light is proportional to 1 / {1+ (1 / m)} ² when the image magnification is m, and therefore decreases as the image is reduced. On the other hand, in order to increase the number of DNA samples to be processed at one time, it is necessary to increase the number of migration paths. If a large number of migration paths are to be secured on one migration plate, the width will increase. On the other hand, the effective diameter of a line sensor or a two-dimensional sensor with an image amplification tube is at most 25 mm, and when attempting to measure a long fluorescent line image, the image magnification m becomes small and the amount of light received decreases.

In the photodetection electrophoretic device of the present invention, a long fluorescent image is divided and detected as a two-dimensional image, so that there is an advantage that the image magnification m need not be reduced. Since the gel plate is a laminated type, gels can be arranged at regular intervals, and the number of gel plates can be increased if necessary. A glass plate with a thickness of 5 mm is usually placed between the gels.
Even if the gels are arranged side by side, the thickness is about 5 cm or more, and the measurement can be performed without any trouble by using a two-dimensional detector. Further, since the holding container for the photodetection electrophoretic device of the present invention has the above-mentioned configuration, it is possible to easily prepare a large number of gel plates by injecting the gel material into the holding container for housing the gel holding plate. You can

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION A reference example will be described below with reference to FIG. 1, and an embodiment of the present invention will be described with reference to FIGS.
In FIG. 1, the four electrophoretic plates 4, ..., 4 are respectively
A gel holding plate made of 6% polyacrylamide was formed in a gap of 0.3 mm between two gel holding plates consisting of 300 mm × 200 mm × 5 mm quartz plates, and a buffer tank and a power source were DNA analysis equipment manufactured by Atto. Using. In the upper part of the gel plate between the gel holding plates in each migration plate, 30 sample injection grooves (2 mm width, center interval 4
mm) are formed by comb combs. Each migration plate 4 is provided with a buffer standing on the lower buffer tank 6,
An upper buffer tank 5 is provided on one side of each electrophoresis plate 4 separately.
Are fixed, and the buffer in the upper buffer tank 5 is supplied to the upper part of each gel plate through an opening on the side surface of the upper buffer tank 5 (not shown). A cathode and an anode are respectively installed in the upper buffer tank 5 and the lower buffer tank 6 as shown in the figure, and the DNA fragment migrates downward by the voltage of 1.2 KV applied between the both electrodes.

The fluorescently labeled DNA fragment fragmented for nucleotide sequence determination is injected onto the upper part of the electrophoretic plate. 2 from the migration start point
The position of 5 cm inside and outside is uniformly irradiated with a laser from the side of the gel, and the fluorophores (FITC; 4) that migrate on four different types of terminal bases of four types of adenine, cytosine, guanine, and thymine. When a DNA fragment labeled with fluoresceine isothiocyanate (emission maximum 515 nm) passes therethrough, it fluoresces. When viewed from the bottom, lines that emit fluorescence as many as the migration plates are observed. These line images are formed by a lens 7 with a filter on a two-dimensional detector 8 equipped with an image amplifier or a highly sensitive two-dimensional sensor.

In this example, since there are four gel plates, the line image corresponding to this is four lines 13 as displayed on the monitor 12. It can be seen that the fluorescence intensity in each line changes with time as shown in FIG. 2, and the fluorescence intensity at the portion corresponding to the migration path of the sample changes. A two-dimensional sensor can be viewed as a collection of many line sensors, but the information of four line images is obtained by processing the signal from each corresponding line sensor.

Further, four types of bases are labeled with fluorescent substances having different wavelengths, and a prism or the like is inserted before or after the condenser lens 7 (not shown) to disperse the fluorescence line image 13 in the vertical direction. It is also possible to process the line images from the portions corresponding to the respective wavelengths of the above four types of fluorescent dyes to determine the DNA base sequence. That is, in this fluorescence, the time changes of the respective maximum positions of the fluorescence corresponding to the four kinds of terminal base species are as shown in FIG. 3, and the time taken for the DNA fragment group of each terminal base species to pass through the light irradiation part is shown. Recognize. In FIG. 3, the symbols A, C, G, and T represent adenine, cytosine, and
Represents the four types of terminal bases of guanine and thymine,
Curves of a solid line, a dotted line, a one-dot chain line, and a two-dot chain line in the figure are curves showing temporal intensity changes of light having wavelengths corresponding to A, C, G, and T, respectively. From now on D
The NA base sequence can be determined. The number of migration paths can be quadrupled in the embodiment as compared with the case of one gel plate, but the number of gel plates can be 10 or more.

Next, an embodiment of the present invention will be described with reference to FIGS. In FIG. 4, a migration plate holding frame 21 (internal dimension 300 m, which constitutes a holding container for a photodetection electrophoresis device)
m × 120 mm × 38 mm) is made of a transparent acrylic resin, and an unpolymerized gel material injection port 24 is provided under the transparent acrylic resin. A communication groove (not shown) is provided on the upper surface of the bottom lid 25.

First, a glass plate 22 (250 mm × 120 mm × 5) as a gel holding plate is provided in the migration plate holding frame 21.
mm) are arranged at intervals of 0.3 mm in the frame, and the injection port 2
When the unpolymerized material of 4 to 4.5% polyacrylamide gel is injected, the unpolymerized material flows into the space between the glass plates 22 through the communication groove on the upper surface of the bottom lid 25 to be filled, and then the upper surface of the gel plate 23. 30 sample injection grooves (2 mm width, center interval 4 mm, not shown) are formed with a comb-shaped comb and left at room temperature to solidify the unpolymerized material, thereby gelling between the glass plates 22. The plate 23 is formed.

FIG. 5 is a view of the laminated gel thus produced, viewed from above. When the number of gels is small,
Insert a spacer 26 to prevent the gel from entering in unnecessary places. The upper part of the holding frame is open so that it can be filled with the buffer solution. If an electrode is attached and an upper lid (not shown) having a gas vent hole is fitted on the upper part of the holding frame, the upper space of the holding frame can be used as an upper buffer solution tank during migration. The prepared laminated gel is set in the lower buffer tank 28.

On the other hand, the fluorescence labeled DNA fragment is prepared in the same manner as in the case of the reference example of FIG. 1 and injected into the sample injection groove on the upper surface of the gel plate. Then, by applying a voltage of 1.0 KV between the upper and lower buffer solution baths, the fluorescence-labeled DNA fragment migrates downward, and the fluorescence detection from the DNA fragment is detected by the measurement system of the photodetection electrophoresis apparatus shown in FIG. Do. Argon laser 29 (488nm, 10mW)
The light emitted from is reflected by a beam splitter 30 composed of a half mirror and a total reflection mirror, and irradiates the gel plate 23 from the side surface.

Of course, it is also possible to irradiate in the buffer solution near the end of the gel and to irradiate the DNA fragment that has come out of the gel. In this case, for example, in the operation of forming the gel plate 23, a spacer having a required height protruding from the upper surface of the bottom pig is fitted in advance to the lower end of the gap of the glass plate 22 in which the gel plate is formed. In the same manner as above, the gel plate 23 is formed by filling the gap of the glass plate 22 where the gel plate is formed with an unpolymerized material. After that, the bottom lid is replaced with the one not provided with the spacer so as to be fitted, and the lower end of the glass plate 22 on which the gel plate is not formed is irradiated with laser. By doing so, the refraction of the light by the gel is eliminated, so that the laser light can be reflected by the mirror without being split by the mirror to irradiate directly under all the gel end portions.

When viewed from the lower end side of the laminated gel, the linear portions emitting fluorescence are lined up in front and back. This fluorescent image is formed by a lens 31 with a filter on a high-sensitivity two-dimensional sensor 32 (manufactured by Hamamatsu Photonics KK) and detected. When there are three gel plates, three fluorescent images 38 can be observed as shown on the television monitor 36.

[0025]

According to the photodetection electrophoretic device of the present invention,
Information from a large number of electrophoretic plates can be obtained at the same time with one measuring device, and the electrophoretic paths for processing the sample are increased in proportion to the number of the gel plates to improve the processing capability of fluorescence detection. Fluorescence detection of a sample, for example, sequencing of a large number of DNA fragments such as analysis of human genes can be rapidly performed.

[Brief description of drawings]

FIG. 1 is a conceptual diagram of a reference example of a photodetection electrophoresis device.

FIG. 2 is a diagram showing a temporal change in monitor signal intensity by the apparatus of FIG.

FIG. 3 is a view showing a time change of light intensity of four kinds of wavelengths corresponding to four kinds of terminal base species of a DNA fragment.

FIG. 4 is a schematic view showing an example of a holding container for a photodetection electrophoresis device.

5 is a plan view of the container of FIG. 4 seen from above.

6 is a conceptual diagram of an example of a photodetection electrophoresis device using the holding container of FIG.

[Explanation of symbols]

1 ... Laser light source, 2 ... Laser light, 3 ... Reflection mirror, 4 ...
Electrophoresis plate, 5 ... Upper buffer tank, 6 ... Lower buffer tank, 7 ... Condensing lens, 8 ... Two-dimensional detector, 9 ... Control device, 10 ... Data processing device, 11 ... Display device, 12 ... Monitor, 13 ... Fluorescence image, 14 ... Horizontal coordinate of migration plate, 1
5 ... Fluorescence intensity, 21 ... Holding frame, 22 ... Glass plate, 23 ...
Gel plate, 24 ... Gel material injection port, 25 ... Bottom pig, 26 ...
Spacer, 27 ... Electrode, 28 ... Lower buffer tank, 29
... laser, 30 ... mirror, 31 ... lens, 32 ... two-dimensional detector, 33 ... controller, 34 ... display device, 35
... data processor, 36 ... monitor, 37 ... buffer solution, 38 ... monitor fluorescence image

Claims (2)

[Claims] 1. A plurality of holding plates laminated substantially parallel to each other with a predetermined gap, and a plurality of plate-shaped electrophoretic members held in the gaps, wherein each of the plurality of plate-shaped electrophoretic members has a plurality of plates. The electrophoretic migration path is set for the laminated electrophoretic medium. 2. An electrophoretic separation unit that electrophoretically separates a fluorescently labeled sample, a unit that applies a voltage to the electrophoretic separation unit, a unit that optically excites the sample separated by the electrophoretic separation unit to generate fluorescence, A photodetection electrophoretic device including a means for detecting the photoelectrophoresis device, wherein the electrophoretic separation unit includes the stacked electrophoretic medium according to claim 1.