JPH08211017A - Dna base sequence determining apparatus - Google Patents

Abstract

(57) [Abstract] [Purpose] To provide an electrophoresis apparatus for simultaneous analysis of various samples. A plurality of electrophoretic paths through which a DNA fragment labeled with a phosphor migrates, a light irradiating means for irradiating the electrophoretic path with a laser beam, and a photodetector for detecting fluorescence emitted from the phosphor by the irradiation of the laser beam. In the DNA base sequence determination device for detecting a DNA fragment, the light irradiation means 1 to 4 include scanning means 2, 3 and 4 for scanning the laser beam 1 and irradiating a plurality of electrophoresis paths. The light detecting means 8 detects the fluorescence from the fluorescent substance that labels the DNA fragment that migrates in each of the plurality of electrophoretic paths in synchronization with the scanning of the laser light, and thus the DNA that migrates in each electrophoretic path. The terminal base species of the fragment is identified, and the base sequence of the nucleic acid sample is determined. [Effect] The distance between migration lanes can be reduced, and the difference in migration conditions between migration 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 electrophoretic device, and more particularly to a DNA base sequencer (DNA sequencer).
The present invention relates to an electrophoretic device suitable for use as.

[0002]

2. Description of the Related Art Conventionally, a method using a radioisotope has been used to determine a base sequence on DNA, but it is desired to develop a method for optically detecting a base by fluorescent labeling. 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.
(Fiscal 1983 Grant-in-Aid for Scientific Research (Comprehensive Research (A)) Research Results Report P.20-25)
A method in which a group of seed fragments is labeled with phosphors having different fluorescence wavelengths, migrated in a column-shaped gel, and the light emitted by light irradiation is dispersed by a diffraction grating and detected by four photomultiplier tubes. Is also proposed.

[0003]

[Problems to be Solved by the Invention] In these proposed methods, it is necessary to provide a photodetector for each DNA fragment group, and four detectors are required for one type of sample. Although a photomultiplier tube is used as the photodetector, when a flat plate type electrophoresis plate is used, the photomultiplier tubes are arranged in a straight line on each migration path. Therefore, the migration path interval is determined by the size of the photomultiplier tube, and 10 mm is the minimum. Therefore, analysis of one DNA sample requires four migration zones 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 zones at once. However, as described above, according to the conventional method, it was possible to measure at most 20 electrophoretic bands on one electrophoretic plate (effective width of up to 20 cm) and to measure 5 samples. That is, there is a drawback that the ability to simultaneously analyze various samples is lacking.

[0005]

The present invention has been made to solve the above problems. In the present invention, one detector is installed in a plurality of electrophoretic zones instead of providing a detector for each electrophoretic zone in the related art, and the light from each electrophoretic zone is detected by time division. 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. As means for time-division and detection of light from each migration zone, each migration path is time-divided with light, or each migration path is always irradiated with light. A light-shielding member is provided between them so that light from each migration path is selectively transmitted to the detector.

[0006]

[Function] One detector is installed in a plurality of electrophoretic zones, and the light from each electrophoretic zone is time-divisionally detected to detect the number of electrophoretic zones more than the number of detectors. Therefore, it is possible to increase the number of electrophoretic bands installed, which was limited by the installation location of the detector.

[0007]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to FIG. The laser beam 1 is reflected by the rotating mirror 4 and the like, and then reflected by the mirrors 2-3, 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 path 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 respectively irradiate the migration paths of the fragment groups whose ends are stopped at T, G, and C. Although the irradiation optical path is changed by operating the rotating mirror 4 in this way, it is possible to irradiate a plurality of migration paths made up of four sets by dividing by a half mirror or the like.

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. The fluorescence signals from the four migration paths are alternately input to the detector 8, and the signals for each migration path are integrated by using the synchronization circuit 11 so that the signals are integrated so as not to mix.
To store. Considering a two-dimensional memory, the horizontal axis indicates the type of migration zone, and the vertical axis indicates time or sampling number. You can take The time change of the fluorescence intensity from each migration zone can be known by reading the column of memory. If there is a difference in measurement time between the four migration zones, and this is a problem, the rotation cycle of the rotating mirror may be shortened, but a cycle 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 represents time, and (a) is a signal 15 'that appears for each migration zone. (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 rotating mirror is used to deflect the light beam. However, the same operation can be performed by scanning the beam or using a mirror which moves the piston.

In the above embodiment, since the laser light is divided and used, the amount of irradiation light per lane is small. FIG. 3 shows an embodiment made to solve this difficulty. The laser light 1 is incident on the gel from the side surface and irradiates a fixed part of the entire migration zone. Although fluorescence is emitted from each migration zone, only fluorescence in a specific lane can pass through the slit 18 and reach the detector. Driver is a slit 19
With this, it is possible to move to the left and right, and it is possible to pass the fluorescence signal from different migration zones at regular intervals. Signals are integrated and data processed in synchronization with the movement of the slit. In this example, detectors may be provided on both sides of the electrophoretic panel, and the slits may be opened and closed in two steps to increase the measurement time per electrophoretic zone.

[0012]

As described above, according to the present invention, the migration lane interval can be made smaller than that specified by a photomultiplier tube or the like, which is suitable for the analysis of various samples requiring many migration lanes. It is valid. Further, there is an advantage that the difference in the migration condition between migration lanes due to temperature unevenness can be reduced by reducing the migration lane interval.

[Brief description of drawings]

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

FIG. 2 is a diagram showing a time change of a detection signal, (a) a time change of a fluorescence signal arranged for each migration zone, (b) a time change of a fluorescence signal intensity entering a detector, and (c). 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 ... Reflecting 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 detector, 17' ... Intermittent light pulse, 18 ... Movable slit, 19 ... Driver, 20 ... Photodetector, 21 ... DNA Fragment group, 22 ... Migration direction, 23 ...
Electrophoretic DNA band.

Claims (7)

[Claims] 1. A DNA fragment prepared from a nucleic acid sample, wherein a plurality of electrophoretic paths along which fluorescently labeled DNA fragments migrate, and a light irradiation means for irradiating the electrophoretic path with laser light are provided. A DNA base sequence determination device for detecting the fluorescence emitted from the phosphor upon irradiation of laser light, wherein the light irradiation means scans the laser light, Scanning means for irradiating an electrophoretic path is provided, and the photodetecting means scans the laser light with the fluorescence from the fluorescent substance that labels the DNA fragment that migrates in each of the plurality of electrophoretic paths. DNA base sequence determination, characterized in that the terminal base species of the DNA fragments that migrate in each of the electrophoretic paths are identified by synchronous detection, and the base sequence of the nucleic acid sample is determined. Location. 2. The end base species of the DNA fragment and the DNA fragment of the DNA fragment are made to correspond by correlating the time point when the fluorescence is detected by the light detecting means and the electrophoretic path at the position irradiated by the scanning of the laser beam. The DN according to claim 1, wherein the length is determined and the base sequence of the nucleic acid sample is determined.
A base sequencer. 3. The scanning means includes a light reflecting means for changing the scanning direction of the laser light, and a light splitting means for splitting the laser light into light traveling in mutually different directions. Item 1. A DNA nucleotide sequencer according to item 1. 4. The DNA base sequencer according to claim 1, wherein the fluorescence is detected by separating each of the electrophoresis paths by time division. 5. The fluorescence is detected by making the time when the fluorescence is detected by the light detecting means and the electrophoretic path irradiated by the scanning of the laser light, and separating the electrophoretic paths by time division. The DNA nucleotide sequencer according to claim 1, wherein 6. A plurality of electrophoretic paths along which a DNA fragment labeled with a fluorescent substance migrates, a light irradiating means for irradiating the electrophoretic path with a laser beam, and fluorescence emitted from the fluorescent substance by the irradiation with the laser beam. In the DNA base sequence determination device for detecting the DNA fragment, the light detecting means corresponds to each group in which the plurality of electrophoresis paths are divided into a plurality of groups. Each of the photodetectors provided corresponding to each of the groups includes an provided photodetector, and each of the photodetectors is time-divided from the DNA fragments that migrate in each of the electrophoretic paths in each of the groups. An apparatus for determining a DNA base sequence, which is characterized in that: 7. A plurality of electrophoretic paths along which a DNA fragment labeled with a fluorescent substance migrates, a light irradiation means for irradiating the electrophoretic path with a laser beam, and a fluorescence emitted from the fluorescent substance by the irradiation with the laser beam. In a DNA base sequence determination device for detecting the sample fragment, comprising:
The light irradiation means has a scanning means for scanning the laser light, and the light detection means is a photodetector provided corresponding to each group in which the plurality of electrophoresis paths are divided into a plurality of groups. The photodetector provided corresponding to each of the groups, the fluorescence from the sample fragment that migrates in each of the electrophoretic paths in each of the groups, in synchronization with the scanning of the laser light. A DNA nucleotide sequencer characterized by detecting.