JP2666799B2 - Electrophoresis device - Google Patents

Description

Description: 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).
The present invention relates to an electrophoresis apparatus suitable for use as a device. 2. Description of the Related Art Conventionally, a method using a radioisotope has been used to determine a base sequence on DNA. However, it has been desired to develop a method for optically detecting a base by fluorescently labeling the base. I have. 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] The present invention has been made to solve the above problems. In the present invention, the conventional 1
Instead of providing a detector for each migration band, one detector is provided for a plurality of migration bands, and light from each migration band is detected in a time-sharing 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. A detector is provided for a plurality of migration bands, and the light from each migration band is detected in a time-division manner. The number of migration bands is larger than the number of detectors. Therefore, the number of migration bands, which is limited by the location of the detector, can be increased. 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. 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 effective for analysis. 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. [Description of Signs] 1 ... Laser light, 2 ... Partial reflection plate, 3 ... Reflection mirror, 4 ... Rotating mirror, 5 ... Electrophoresis glass panel, 6 ... Electrophoresis gel, 7 ... Filter, 8 ... Photomultiplier tube, 9 ... Detection circuit, 10 ... Integrator, 11 ... Synchronization 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.

Continuation of front page    (56) References JP-A-61-213748 (JP, A)                 JP-A-61-155732 (JP, A)                 JP-A-56-61633 (JP, A)                 JP-A-61-62843 (JP, A)                 JP-A-51-13298 (JP, A)                 JP-A-51-63673 (JP, A)                 JP-A-53-33189 (JP, A)                 JP-A-57-204437 (JP, A)                 JP-A-63-36147 (JP, A)                 Actual Opening Showa 49-20582 (JP, U)                 Tokiko Sho 53-21408 (JP, B1)

Claims (1)

(57) [Claims] A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates; a light irradiation unit configured to irradiate the plurality of electrophoretic paths with laser light; and detecting fluorescence emitted from the fluorescent substance by the irradiation of the laser light. In the electrophoresis apparatus having a light detection unit, the light irradiation unit includes a unit that scans the laser beam and irradiates each of the plurality of electrophoresis paths, and the light detection unit includes An electrophoresis apparatus, wherein the fluorescence from the nucleic acid sample migrating the illuminated electrophoresis path is detected for each of the plurality of electrophoresis paths. 2. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates; a light irradiation unit configured to irradiate the plurality of electrophoretic paths with laser light; and detecting fluorescence emitted from the fluorescent substance by the irradiation of the laser light. In the electrophoresis apparatus having a light detection unit, the light irradiation unit includes a unit that scans the laser beam and irradiates each of the plurality of electrophoresis paths, and the light detection unit includes An electrophoresis apparatus, wherein a time change in the intensity of the fluorescence from the nucleic acid sample being electrophoresed on the illuminated electrophoresis path is detected for each of the plurality of electrophoresis paths. 3. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates; a light irradiation unit configured to irradiate the plurality of electrophoretic paths with laser light; and detecting fluorescence emitted from the fluorescent substance by the irradiation of the laser light. In the electrophoresis apparatus having light detection means, the plurality of electrophoresis paths are divided into a plurality of groups including a plurality of electrophoresis paths, and the light detection means includes a plurality of electrophoresis paths of each of the groups. An electrophoresis apparatus for detecting the fluorescence from the nucleic acid sample during electrophoresis. 4. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates; a light irradiation unit configured to irradiate the plurality of electrophoretic paths with laser light; and detecting fluorescence emitted from the fluorescent substance by the irradiation of the laser light. In an electrophoresis apparatus having a light detection unit, the light irradiation unit includes a unit configured to scan the laser beam and irradiate each of the plurality of electrophoresis paths, and the plurality of electrophoresis paths include a plurality of electrophoresis paths. Electrophoresis, wherein the photodetection means detects the fluorescence from the nucleic acid sample during the electrophoresis of each of the electrophoresis paths in each of the groups. apparatus. 5. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates; a light irradiation unit configured to irradiate the plurality of electrophoretic paths with laser light; and detecting fluorescence emitted from the fluorescent substance by the irradiation of the laser light. In an electrophoresis apparatus having a light detection unit, the light irradiation unit includes a unit configured to scan the laser beam and irradiate each of the plurality of electrophoresis paths, and the plurality of electrophoresis paths include a plurality of electrophoresis paths. Divided into a plurality of groups consisting of electrophoresis paths, wherein the light detection means detects a time change in the intensity of the fluorescence from the nucleic acid sample during electrophoresis of each of the electrophoresis paths in each group. Characteristic electrophoresis device. 6. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates; a light irradiation unit configured to irradiate the plurality of electrophoretic paths with laser light; and detecting fluorescence emitted from the fluorescent substance by the irradiation of the laser light. In an electrophoresis apparatus having a light detection unit, the light irradiation unit includes a unit configured to scan the laser beam and irradiate each of the plurality of electrophoresis paths, and the plurality of electrophoresis paths include a plurality of electrophoresis paths. Divided into a plurality of groups consisting of electrophoresis paths, the light detection means has a light detector provided corresponding to each of the groups, the light detector provided corresponding to each group, An electrophoresis apparatus, wherein the fluorescence from the nucleic acid sample is electrophoresing each of the electrophoresis paths in each of the groups. 7. The electrophoretic device according to claim 6, wherein the laser light is applied to each of the plurality of electrophoresis paths at a position at a predetermined distance from a migration start point of the plurality of electrophoresis paths. Electrophoresis device. 8. 7. The electrophoretic device according to claim 6, wherein the light irradiating unit includes: a light reflecting unit that changes a scanning direction of the laser light; and a light dividing unit that divides the laser light into light traveling in different directions. An electrophoresis apparatus comprising: 9. 7. The electrophoresis apparatus according to claim 6, wherein the fluorescence is detected for each electrophoresis path of each group in synchronization with the scanning of the laser light. 10. 7. The electrophoresis apparatus according to claim 6, wherein each group includes four electrophoresis paths. 11. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates; a light irradiation unit configured to irradiate the plurality of electrophoretic paths with laser light; and detecting fluorescence emitted from the fluorescent substance by the irradiation of the laser light. In an electrophoresis apparatus having a light detection unit, the light irradiation unit includes a unit configured to scan the laser beam and irradiate each of the plurality of electrophoresis paths, and the plurality of electrophoresis paths include a plurality of electrophoresis paths. Divided into a plurality of groups consisting of electrophoresis paths, the light detection means has a light detector provided corresponding to each of the groups, the light detector provided corresponding to each group, An electrophoresis apparatus, wherein a change over time in the intensity of the fluorescence from the nucleic acid sample during electrophoresis in each of the electrophoresis paths in each of the groups is detected. 12. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates; a light irradiation unit configured to irradiate the plurality of electrophoretic paths with laser light; and detecting fluorescence emitted from the fluorescent substance by the irradiation of the laser light. In the electrophoresis apparatus having light detection means, the light irradiation means substantially applies the laser light to the plurality of electrophoresis paths at a position at a predetermined distance from a migration start point of the plurality of electrophoresis paths. Simultaneously irradiate, the plurality of electrophoresis paths are divided into a plurality of groups consisting of a plurality of electrophoresis paths, and the light detection means is the nucleic acid sample which is migrating each of the electrophoresis paths of each group. An electrophoresis apparatus characterized by detecting the fluorescence from a target. 13. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates; a light irradiation unit configured to irradiate the plurality of electrophoretic paths with laser light; and detecting fluorescence emitted from the fluorescent substance by the irradiation of the laser light. In the electrophoresis apparatus having light detection means, the light irradiation means substantially applies the laser light to the plurality of electrophoresis paths at a position at a predetermined distance from a migration start point of the plurality of electrophoresis paths. Simultaneously irradiate, the plurality of electrophoresis paths are divided into a plurality of groups consisting of a plurality of electrophoresis paths, and the light detection means is the nucleic acid sample which is migrating each of the electrophoresis paths of each group. An electrophoresis apparatus, which detects a time-dependent change in the intensity of the fluorescence from the object. 14． A plurality of electrophoresis paths on which a nucleic acid sample labeled with a fluorescent substance migrates; a light irradiation unit configured to irradiate the plurality of electrophoretic paths with laser light; and detecting fluorescence emitted from the fluorescent substance by the irradiation of the laser light. In the electrophoresis apparatus having light detection means, the light irradiation means substantially applies the laser light to the plurality of electrophoresis paths at a position at a predetermined distance from a migration start point of the plurality of electrophoresis paths. Simultaneously irradiate, the plurality of electrophoresis paths are divided into a plurality of groups consisting of a plurality of electrophoresis paths, and the light detection means has a photodetector provided corresponding to each of the groups, An electrophoresis apparatus, wherein the photodetectors provided for each group detect the fluorescence from the nucleic acid sample migrating through each of the electrophoresis paths in each group. 15. 15. The electrophoresis apparatus according to claim 14, wherein the plurality of electrophoresis paths move between the plurality of electrophoresis paths and the photodetectors provided corresponding to the respective groups in a direction intersecting the plurality of electrophoresis paths. And a slit for allowing the fluorescence from each of the electrophoresis paths to pass therethrough. 16. 15. The electrophoresis apparatus according to claim 14, wherein the plurality of electrophoresis paths move between the plurality of electrophoresis paths and the photodetectors provided corresponding to the respective groups in a direction intersecting the plurality of electrophoresis paths. And a slit for passing the fluorescence from each of the electrophoresis paths, and the photodetector operates in synchronization with the movement of the slit. 17． 15. The electrophoresis apparatus according to claim 14, wherein each group includes four electrophoresis paths. 18. A plurality of electrophoresis paths on which a nucleic acid sample labeled with a phosphor migrates, a light irradiation unit for irradiating the plurality of electrophoresis paths with a laser beam, and detecting fluorescence emitted from the phosphor by the irradiation of the laser beam In an electrophoresis apparatus having a light detection unit, the light irradiation unit substantially applies the laser light to the plurality of electrophoresis paths at a position at a predetermined distance from a migration start point of the plurality of electrophoresis paths. Simultaneously irradiate, the plurality of electrophoresis paths are divided into a plurality of groups of a plurality of electrophoresis paths, and the light detection means has a photodetector provided corresponding to each of the groups, The light detector provided for each group detects a time change in the intensity of the fluorescence from the nucleic acid sample during electrophoresis of each of the electrophoresis paths of each group. Electrophoresis device.