JPH01299462A - Gene detector - Google Patents

Abstract

PURPOSE:To perform quantitative analysis by simple handling, by marking DNA probes with fluorescent light, hybridizing the probes with objective genes, thereafter separating unbonded probes with gel, and measuring the bonded DNA probes optically. CONSTITUTION:Cells 2 which are fixed to a holding plate 1 are filled with gel 3. Specimen is injected on the upper part of each cell. The cells 2 are immersed in upper and lower buffer liquids 4. When a voltage is applied across electrodes 5 and 5', the DNA specimen moves downward. The single body of the DNA probe flows into the lower buffer liquid 4. Meanwhile, the DNA which is marked with the fluorescent light and hybridized with the objective gene is hardly moved. Therefore, only the DNA probe which is not hybridized is extracted. Light which is emitted from a light source 6 is introduced from the upper part of each cell 2 and projected on the tube shaped gel. The fluorescent light which is emitted from the hybridized DNA probe in the gel is inputted into a photodetector 9 through a lens system 7. The output from the photodetector 9 is inputted into a detector circuit and a data processing part 10. Thus the quantitative analysis data are obtained.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a device for detecting genes such as DNA or RNA, and particularly to a device for detecting genes using fluorescent probes capable of rapid detection with good quantitative properties. .

[Conventional technology]

For testing for genetic diseases, etc., DNA complementary to the gene causing the disease is synthesized and used as a DNA probe. The test gene is immobilized on a cellulose or Suilon filter, and a radioactive am or dye-labeled DNA probe is sprinkled on it to hybridize (bond) with the DNA on the filter. Since the dye-labeled DNA is selectively captured, it is detected by autoradiography or visual inspection to determine whether the target disease-causing gene is present. Regarding such conventional technology, please refer to
1986, pages 462 to 468.

[Problem to be solved by the invention]

The above-mentioned conventional techniques require a lot of effort and time, and also have problems in quantitative performance.

The purpose of the present invention is to overcome these problems and provide a method that is easy to handle and has quantitative properties.

[Means to solve the problem]

The above purpose is to fluorescently label a DNA probe, hybridize it with the target gene, use a gel to separate the DNA probe bound to the target gene from the unbound probe, and optically measure the bound DNA probe. This is achieved by

[Effect]

By using a fluorescently labeled probe, there is no need to use radioisotopes, and since the fluorescence from the DNA probe attached to the target gene remaining in the gel is detected, the hassle of transferring it to a filter is eliminated, increasing operational efficiency. goes up.

〔Example〕

An embodiment of the present invention will be described below with reference to FIG.

FIG. 1 shows the sample separation cell 2 and cell holding plate 1 viewed from above, and the sample cells 2 are lined up in a row. FIG. 2 is a sectional view of an example of the configuration of an apparatus for electrophoretically separating a sample.

A gel 3 is filled in a cell 2 fixed to a holding plate 1. The sample is injected onto the top of gel 3. The cell 2 will be immersed in the upper and lower buffer solutions 4. When a voltage (usually around 50 V/cxm) is applied between electrodes 5 and 5', D
The NA sample moves from top to bottom. The migration speed of DNA varies depending on the base length N, the electrophoretic field E, and the temperature T.

FIG. 4 shows the time required for single-stranded DNA of various lengths to migrate at 25 am. Straight line 17°18.1
9 has a polyacrylamide gel concentration of 2% and 4%, respectively.
and 6%. The length of the DNA probe is about 20 bases, and the length of the target DNA is 1000 or more bases. Even short ones are about 300 to 400. Therefore, as a representative example, consider a case where the target genetic predictor length is 1000.

In the example, the length of the gel portion in the cell is 2511IIl.

The concentration of polyacrylamide gel was 4%. A sample of the gene to be tested sprinkled with a DNA probe is injected from the top of the gel, and when electrophoresis begins, the DNA probe alone flows out into the lower buffer tank after about 2 minutes. Outflow can be confirmed by detecting fluorescence from the probe. On the other hand, the DNA hybridized with the target gene passes through the gel part only after 30 minutes have passed. Therefore, the electrophoresis electric field is applied for about 5 minutes to remove unhybridized DNA.
Only the probe is removed, and the cell 3 is moved to the detection section with the cell 3 attached to the holding plate. An example of the configuration of the detection section is shown in FIG. Light 11 emitted from a light source 6 such as a laser or a lamp enters the cell 2 from above and irradiates the tubular gel. Fluorescence 20 is observed when there is a hybridized DNA probe in the gel. Fluorescent light 20 is collected by a lens 7, passes through a filter 8, enters a light receiving element 9, and is detected by a detector 10. The cells 2 arranged on a straight line are sequentially sent to the measuring section by an automatic feeding mechanism and measured.

Of course, instead of moving the cell, the light source and detector can also be moved.

Although different cells were used for each sample in the above embodiments, plate-shaped gels may also be used. FIG. 5 is an example. A sample is injected into the groove of the gel, and a plurality of cells 1 are injected into the gel plate 3.
2, extract the free DNA probe in the same manner as in the previous example, irradiate it with light 11, and measure the fluorescence.

FIG. 6 shows an example in which the plate-shaped gel 3 is irradiated with the laser 11 from the lateral direction. In this example, by irradiating and scanning all cells simultaneously, the highly sensitive line sensor 15 can detect information from a plurality of cells simultaneously. When processing a larger amount of samples, a plate with cells distributed on a plane as shown in FIG. 7 can also be used. In this case, detection may be performed for each cell, for each line, or by irradiating the entire surface with light and using a two-dimensional inspection device.

In the examples so far, separation and measurement are performed separately, but the device configuration can also be combined into one.

〔Effect of the invention〕

According to the present invention, without using a radioisotope label,
The target gene can be detected in a short time without the need to immobilize the target DNA on a filter. Another advantage is that the fluorescence intensity can be measured quantitatively.

[Brief explanation of the drawing]

FIG. 1 is a plan view of a cell section according to an embodiment of the present invention, FIG. 2 is a sectional view of an apparatus for electrophoretic separation according to an embodiment of the present invention, and FIG. 3 is a fluorescence measurement according to an embodiment of the present invention. Fig. 4 is a graph showing the relationship between the length of DNA bases and migration time, and Fig. 5 is an embodiment of the present invention in which each cell is irradiated with light using a plate-shaped gel. Fig. 6 is a block diagram of an embodiment of the present invention in which light is incident from the side and all cells are simultaneously irradiated and measured, and Fig. 7 is a schematic diagram of the structure of two-dimensionally distributed cells. . DESCRIPTION OF SYMBOLS 1... Cell holding plate, 2... Sample cell, 3... Gel, 4... Buffer solution, 5,5'... Electrode, 6...
・Light source, 7... Lens, 8... Filter, 9...
・Photodetector, 1o...detection circuit and data processing section,
DESCRIPTION OF SYMBOLS 11... Irradiation light, 12... Sample cell, 13... Gel holder, 14... Filter, lens system, 15...
・Line sensor or two-dimensional sensor, 16...
Data processing machine, 17... Figure 4 Figure 6 Figure 7

Claims (1)

[Claims] 1. In an apparatus for detecting a specific gene by an optical method, a free DNA probe and a DNA probe hybridized to a subject gene are separated using a gel, and the former is drained into a buffer solution. A gene detection device characterized in that the latter is retained in a gel and the latter or the former is detected. 2. The gene detection device according to claim 1, wherein the separation gel is held in a short cylindrical member. 3. The gene detection device according to claim 1, wherein the separation gel is constituted by a rod-shaped gel, and a plurality of wells for sample injection are provided above the gel. 4. The gene detection device according to claim 1, wherein the separation gel is composed of a flat gel, and the sample injection wells are distributed two-dimensionally. 5. A gene detection device according to any one of claims 1 to 4, characterized in that it has a mechanism for sequentially sending gel portions or separated cells holding genes to a light irradiation section. 6. The device according to any one of claims 1 and 3 to 4, characterized by having the function of simultaneously irradiating a plurality of gene holding regions arranged vertically with a laser and sequentially shifting them in the horizontal direction. Gene detection device.