JPH1090223A - Electrophoresis device - Google Patents

Abstract

(57) [Summary] [Object] To provide an electrophoresis apparatus for simultaneously analyzing multiple samples. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates, light irradiation means for irradiating the plurality of electrophoretic paths with a laser beam, and detecting fluorescence emitted from the fluorescent substance by the laser beam irradiation In an electrophoresis apparatus having a light detecting means, the light irradiating means includes means for scanning a laser beam to irradiate each of a plurality of electrophoretic paths, and a light detecting means. In this method, fluorescence from a DNA fragment migrating in an electrophoresis path irradiated with laser light is separated for each electrophoresis path in synchronization with the scanning of the laser light, and is repeatedly detected at predetermined time intervals. [Effect] The distance between the electrophoresis lanes can be reduced, and the difference in electrophoretic conditions between electrophoresis lanes due to uneven temperature can be reduced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoresis apparatus, and more particularly to an apparatus for determining a base sequence on DNA (DNA sequencer).
About.

[0002]

2. Description of the Related Art Conventionally, a method using a radioisotope has been used for determination of a base sequence on DNA. However, it is desired to develop a method for optically detecting a base by fluorescently labeling the base. To achieve this, as shown in FIG. 4, a DNA fragment having a specific end and one end being an adenine base (A), a guanine base (G), a cytosine base (C), or a thymine base (T) Have been proposed in which electrophoresis is performed on separate electrophoresis paths, and light is irradiated to measure electrophoretic DNA fragments using photomultiplier tubes provided for each electrophoresis path.
(Scientific Research Grant-in-Aid for Grants-in-Aid for 1983 (Comprehensive Research (A)) Research Report P.20-25)
Species fragment groups are labeled with phosphors with different fluorescence wavelengths, run in a column-shaped gel, and illuminated with light are separated by a diffraction grating and detected by four photomultiplier tubes. Has also been proposed.

[0003]

In these proposed methods, it is necessary to provide a photodetector for each DNA fragment group, and four detectors are required for one kind of sample. Although a photomultiplier tube is used for the photodetector, when a flat plate type electrophoresis plate is used, the photomultiplier tubes are aligned on each migration path. For this reason, the migration path interval is determined by the size of the photomultiplier tube, and the minimum is 10 mm. Therefore, analysis of one DNA sample requires four migration bands and a width of 4 cm of the migration plate.

[0004] On the other hand, in the shotgun method and the like, which have become widespread as a rapid method for determining a DNA base sequence, a target DNA is shredded and sequence determination of various small fragments is performed at the same time. To do this, it is necessary to be able to utilize many migration bands at once. However, as described above, in the conventional method, it was possible to measure at most 20 migration bands and five samples in number on a single migration plate (effective width: 20 cm). That is, there is a problem that the ability to simultaneously analyze a variety of samples is lacking.

[0005]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems. In the present invention, one detector is provided for a plurality of migration bands instead of providing a detector for each migration band in the related art, and light from each migration band is detected in a time-division manner. The obtained signals are obtained from different migration bands at certain times, but by selectively sampling the signals, the signals from each migration band can be distinguished and their time changes can be recorded. Means for time-division detection of light from each migration band include time-division irradiating light to each migration path, or always irradiating light to each migration path, A light shielding member is provided in between to selectively transmit light from each migration path to the detector. To explain the configuration example of the electrophoresis apparatus of the present invention in more detail, a plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates,
Light irradiation means for irradiating a plurality of electrophoresis paths with laser light,
An electrophoretic device having light detection means for detecting fluorescence emitted from a phosphor by irradiation with laser light, wherein the light irradiation means includes means for scanning the laser light in a direction crossing a plurality of electrophoresis paths; The means is configured to detect fluorescence from a nucleic acid sample migrating in the plurality of electrophoresis paths irradiated with the laser light for each electrophoresis path in synchronization with scanning of the laser light.

[0006]

[Function] A single detector is installed in a plurality of migration bands, and light from each migration band is detected in a time-division manner, so that the number of migration bands can be larger than the number of detectors. Therefore, it is possible to increase the number of electrophoresis bands that have been limited due to the location of the detector.

[0007]

An embodiment of the present invention will be described below with reference to FIG. The laser light 1 is reflected by the mirrors 2 and 3 after being reflected by the rotating mirror 4 and irradiates the electrophoretic plate.
The rotating mirror rotates by 90 ° and stands still for about 1 second at an angle of 45 ° with the incident light. Four light paths can be selected depending on the position of the stationary surface, and these correspond to the irradiation light paths 14 to 17. The irradiation optical path 14 irradiates the migration paths of the fragment groups whose ends are stopped at A, and the irradiation paths 15 to 17 illuminate the migration paths of the fragment groups whose ends are stopped at T, G, and C, respectively. By operating the rotating mirror 4 in this manner, the irradiation optical path is changed. However, the irradiation optical path can be divided by a half mirror or the like and a plurality of sets of four migration paths can be irradiated.

[0008] The fluorescence emitted by the light irradiation passes through the filter 7 and enters the detection zone 8. On the other hand, the excitation light is removed by the filter 7. Fluorescent signals from the four migration paths alternately enter the detector 8, but are integrated using the synchronization circuit 11 so that the signals for each migration path are not mixed and stored in the memory 12.
To store. Considering a two-dimensional memory, the horizontal axis indicates the type of migration band, and the vertical axis indicates time or sampling number. Can be taken. The time change of the fluorescence intensity from each migration band can be known by reading the column of the memory. When there is a difference in measurement time between the four migration bands, and this is a problem, the rotation period of the rotating mirror may be shortened, but a period of 1 to 4 seconds is often sufficient.

FIG. 2 shows an example of the signal thus obtained.
It was shown to. The horizontal axis is time, and (a) is a signal 15 'appearing for each migration band. (B) shows a signal 16 'flowing to the detector, in which AGCT signals are detected in time series.
(C) shows the timing at which the excitation light irradiates each migration lane.

In this embodiment, the light beam is deflected by using a rotating mirror. However, the same operation can be performed by using a mirror that scans a beam or uses a mirror that moves with a piston.

In the above embodiment, since the laser beam is divided and used, there is a problem that the irradiation light amount per lane is reduced. FIG. 3 shows an embodiment for solving this difficulty. The laser beam 1 is incident on the gel from the side, and irradiates a fixed portion of the entire migration band. Fluorescence is emitted from each migration band, but only fluorescence in a specific lane can pass through the slit 18 and reach the detector. The slit is driver 19
To move left and right, and it is possible to pass fluorescent signals from different migration bands at regular intervals. The signals are integrated and data processed in synchronization with the movement of the slit. In this example, detectors are provided on both sides of the electrophoresis panel, and the opening and closing of the slit are performed in two stages, so that the measurement time per electrophoresis band can be increased.

[0012]

As described above, according to the present invention, the spacing between the electrophoresis lanes can be made smaller than that specified by a photomultiplier tube or the like. It is valid. In addition, there is also an advantage that a difference in electrophoretic conditions between electrophoretic lanes due to temperature unevenness or the like can be reduced by reducing the interval between electrophoretic lanes.

[Brief description of the drawings]

FIG. 1 is a schematic plan view of an embodiment of the present invention.

2A and 2B are diagrams showing a time change of a detection signal, wherein FIG. 2A shows a time change of a fluorescence signal arranged for each migration band, FIG. 2B shows a time change of a fluorescence signal intensity entering a detector, and FIG. The figure which shows the time change of the light which irradiates each migration zone.

FIG. 3 is a schematic plan view of a modified example of the present invention.

FIG. 4 is an explanatory diagram showing a state of fragmentation and migration separation of DAN according to the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Laser light, 2 ... Partial reflection plate, 3 ... Reflection mirror, 4 ... Rotation mirror, 5 ... Electrophoresis glass panel, 6 ... Electrophoresis gel, 7 ... Filter, 8 ... Photomultiplier tube, 9 ... Detection circuit, 10 ... Integrator, 11 ... Synchronous circuit, 12 ... Memory, 13 ... Output device,
14 ': DNA to be sequenced, 15': fluorescent signal, 16 ': signal flowing to the detector, 17': intermittent light pulse, 18: movable slit, 19: driver, 20: photodetector, 21: DNA Fragment group, 22 ... electrophoresis direction, 23 ...
Running DNA band.

Claims (10)

[Claims] 1. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates, a light irradiating means for irradiating the plurality of electrophoretic paths with laser light, An electrophoresis device having light detection means for detecting emitted fluorescence, the light irradiation means includes means for scanning the laser light in a direction crossing the plurality of electrophoresis paths, and the light detection means An electrophoresis apparatus, wherein the fluorescence from the nucleic acid sample migrating on the plurality of electrophoresis paths irradiated with laser light is detected for each of the electrophoresis paths in synchronization with the scanning of the laser light. 2. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates, light irradiation means for irradiating the plurality of electrophoresis paths with laser light, An electrophoresis device having light detection means for detecting emitted fluorescence, the light irradiation means includes means for scanning the laser light in a direction crossing the plurality of electrophoresis paths, and the light detection means A change in the intensity of the fluorescence from the nucleic acid sample migrating the plurality of electrophoresis paths irradiated with laser light is detected for each of the electrophoresis paths in synchronization with the scanning of the laser light. Electrophoresis device. 3. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates, light irradiating means for irradiating the plurality of electrophoretic paths with a laser beam, An electrophoretic device having light detecting means for detecting emitted fluorescent light, wherein the light detecting means includes a plurality of light detectors whose number is smaller than the number of the electrophoretic paths. 4. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates, light irradiating means for irradiating the plurality of electrophoretic paths with a laser beam, and irradiating the fluorescent substance with the laser beam. An electrophoretic device having light detecting means for detecting emitted fluorescence, the light irradiating means includes means for repeatedly scanning the laser light to irradiate the plurality of electrophoretic paths, and the light detecting means comprises: An electrophoresis apparatus, comprising: a plurality of photodetectors whose number is smaller than the number of electrophoresis paths. 5. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates, light irradiating means for irradiating the plurality of electrophoretic paths with a laser beam, and irradiating the laser beam with the laser light. An electrophoretic device having light detecting means for detecting emitted fluorescence, comprising: means for selecting the electrophoretic path for irradiating the laser light, wherein the light detecting means comprises: An electrophoresis apparatus, wherein the fluorescence from the nucleic acid sample migrating in the electrophoresis path is detected for each of the electrophoresis paths. 6. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates, light irradiation means for irradiating the plurality of electrophoresis paths with laser light, An electrophoretic device having light detecting means for detecting emitted fluorescence, comprising: means for selecting the electrophoretic path for irradiating the laser light, wherein the light detecting means comprises: A change in the intensity of the fluorescence from the nucleic acid sample that migrates through the electrophoresis path,
An electrophoresis apparatus, wherein the detection is performed for each of the electrophoresis paths. 7. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates, light irradiation means for irradiating the plurality of electrophoresis paths with a laser beam, and irradiation of the fluorescent substance by the laser beam irradiation An electrophoresis apparatus having light detection means for detecting emitted fluorescence, comprising: means for repeatedly selecting the electrophoresis path for irradiating the laser light, and electrophoresing the plurality of electrophoresis paths irradiated with the laser light. An electrophoresis apparatus, wherein the fluorescence from the nucleic acid sample is repeatedly detected for each of the plurality of electrophoresis paths. 8. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates, light irradiating means for irradiating the plurality of electrophoretic paths with a laser beam, and irradiating the laser beam with the laser beam. An electrophoresis apparatus having light detection means for detecting emitted fluorescence, comprising means for scanning the irradiation position of the laser light on the plurality of electrophoresis paths, wherein the light detection means is irradiated with the laser light. An electrophoresis apparatus, wherein the fluorescence from the nucleic acid sample migrating through the plurality of electrophoresis paths is detected for each of the electrophoresis paths. 9. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates, light irradiating means for irradiating the plurality of electrophoretic paths with a laser beam, and irradiating the fluorescent substance with the laser beam. An electrophoresis apparatus having light detection means for detecting emitted fluorescence, comprising: means for scanning an irradiation position of the laser light on the plurality of electrophoresis paths, wherein the light irradiation means irradiates the laser light. Means for selecting an electrophoresis path, wherein the light detection means detects a change in the intensity of the fluorescence from the nucleic acid sample migrating the plurality of electrophoresis paths irradiated with the laser light, An electrophoresis apparatus characterized by detecting each time. 10. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates, light irradiating means for irradiating the plurality of electrophoretic paths with a laser beam, and irradiating the fluorescent substance with the laser beam. An electrophoresis apparatus having light detection means for detecting emitted fluorescence, comprising: means for repeatedly scanning the irradiation position of the laser light on the plurality of electrophoresis paths, wherein the plurality of electrophores irradiated with the laser light are provided. An electrophoresis apparatus, wherein the fluorescence from the nucleic acid sample migrating the path is repeatedly detected for each of the plurality of electrophoresis paths.