JP2675068B2 - DNA detector - Google Patents

Description

TECHNICAL FIELD The present invention relates to a gene diagnosis or gene mapping device, and particularly to a DNA capable of obtaining a DNA pattern in a short time.
It relates to a detection device.

[Conventional technology]

Genetic diagnosis methods are used for diagnosing diseases caused by genes and identifying individuals by genes. The Fingerprint method using genes will be described as an example. In this case, the purpose is to discriminate by utilizing the fact that the gene is different for each individual, and in the case of relatives such as parents and children, the fact that there are many similarities is used for parental discrimination. In human genes, DNA with a short and constant sequence appears repeatedly. However, the frequency and place of repetition vary depending on the individual. So first,
A so-called complementary DNA oligomer bonded to this portion by a hydrogen bond (hybridization) is prepared. This oligomer is labeled with a radioactive element in advance. This is usually called a probe. Double-stranded DNA to be analyzed
Is cut with a restriction enzyme that recognizes and cuts a specific sequence.
The DNA fragment thus obtained is electrophoretically separated using agarose gel. The electrophoretically separated DNA is transferred onto a nylon filter, and the double-stranded DAN is denatured into a single strand and fixed on the filter. On this filter
DNA probe DNA is hybridized. This method is called the Southern blot method. Since the probe DNA is attached to the DNA fragment portion where the complementary repeat sequence is present, this DNA band pattern is transcribed to the film. Although this pattern varies from person to person, the gene band pattern of the same person is constant. In addition, since parents and children have many similar parts, they are used for various kinds of appraisals.

A similar method is used for mapping to find the position of the restriction enzyme on the gene. This type of conventional technique is described in Japanese Forensic Medicine Vol. 41, No. 3, pp. 236-241 (1987).

[Problems to be solved by the invention]

In these conventional techniques, there is a tricky process of copying a DNA fragment on a filter and then transferring a band pattern to the film, which requires time and effort. Further, there is a problem that it is necessary to use a radioactive element.

Also, when using a fluorescent label as a label to optically measure the gel during electrophoresis as used in a DAN sequencer, etc., since agarose is used as the separation gel, scattered light is large and highly sensitive measurement cannot be performed. .

The present invention has been made to solve these problems and to easily obtain a DNA band pattern in a short time.

[Means for solving the problem]

The above-mentioned purpose is a fluorescent labeling of the DNA probe, agarose gel electrophoresis separation in a state of being hybridized with a sample DNA fragment containing a complementary portion, the DNA fragment is allowed to flow out into a buffer solution and irradiated with light, This is achieved by observing the fluorescence emitted from the DNA fragment in real time. That is, a gel that is placed in a buffer solution and has a plurality of migration paths for electrophoretically separating a fluorescently labeled sample, and gels that are placed on both sides sandwiching the gel in the direction in which the sample migrates and impart an electrophoretic force to the sample Voltage applied to the sample, means for irradiating the sample that migrates in the buffer solution along the short surface of the gel on the migration end side where the sample flows out from the gel, and fluorescence emitted from the fluorescent label by light in the buffer solution. Is characterized in that it has a light detection means for detecting with, and the light detection means is arranged so as to face the end face of the gel on the migration end side. [Function] Fluorescently labeled DNA is hybridized and electrophoresed. However, by performing band detection in real time, it is possible to omit complicated processes such as Southern blot and autoradiography. In addition, agarose gel is usually used for separation, but this is cloudy and when irradiated with light, scattered light is strongly detected, which interferes with fluorescence measurement. Here, the DNA fragments were extracted from the agarose gel and then irradiated with a laser to eliminate measurement obstacles due to scattering.

〔Example〕

Hereinafter, an embodiment of the present invention will be described with reference to FIG. The gel plate (agarose plate) 7 is installed in the buffer solution tank,
The buffer solution 10 is filled up to the upper surface of the gel plate 7. DNA
The sample in which the fragment and the fluorescently labeled probe are hybridized is injected into the sample injection well 9 and migrates according to the electric field applied between the electrodes 13 and 13 '. The short DNA reaches the gel cross section 14 in order, but these DNA fragments with the DNA probe flow out from the gel into the buffer solution 10. Laser light 11
Is irradiated with the buffer solution near the gel cross section 14 along the end face 14 to excite the flowing out DNA fragment and obtain a fluorescence signal. The position of the specific sequence on the gene can be known from the migration time. The fluorescence signal is a lens, a filter system 3 and a line sensor with a photoelectric amplifier or a vidicon camera 4.
Detected and sent to the data processor 5. The thickness of the light irradiation part 6 is made sufficiently small so that the DNA fragment group does not pass through a part other than the irradiation part due to diffusion.

The width W due to the diffusion of particles in the liquid is D with D as the diffusion coefficient. Given by The existence of the electric field E, the drift velocity V _d is Are tied together. With this Becomes Where k is Boltzmann factor, T is temperature, e is DN
A has a charge, l is a migration distance. It is better to reduce W in order to detect with good separation. For this purpose, it is necessary to make l small and E large. If the length of the analysis part gel is fixed, it is necessary to make the length from the gel to the light irradiation part as small as possible. In the solution, the migration speed of DNA is about 1 cm / min under the electric field strength of 50 V / cm regardless of the base length.
When the electric field strength in the solution is 5 V / cm, 1 K base DNA is 1 cm.
The spread due to diffusion when electrophoresing is about 0.5 mm at room temperature. Since the width of the laser is usually 0.3 mm or less, it is desirable that the spread due to diffusion after the buffer liquid extraction is 0.3 mm or less. For this purpose, the distance from the gel end face to the light irradiation part should be 5 mm or less.

Further, since the electric field strength is inversely proportional to the cross-sectional area of the portion where the ions flow, it is better that the cross-sectional area, that is, the thickness (because the width of the analysis portion is constant) of the portion that flows out from the gel and reaches the photodetection portion is thinner.

FIG. 2 is a modified example of the separation unit of the embodiment. The migration path is divided by glass or quartz (irradiation section) 15 in order to prevent interference between migration paths due to diffusion of DNA after flowing out from gel 7. In this case, a photoelectron amplifier tube can be used as a detector for each migration path. Further, FIG. 3 shows an example in which the width of the detection portion is forcibly narrowed by quartz or the like so that detection can be performed with high sensitivity even when the agarose gel plate 7 thicker than the laser beam width is used. The same thing can be achieved by reducing the thickness of the gel as it goes to the detection part.

〔The invention's effect〕

According to the present invention, there is an advantage that the DNA pattern information can be processed by a computer without the troublesomeness of using the radioactive element label and the troublesome process such as the filter transcription of the separated DNA. When it is attempted to measure the DNA that is running through the agarose gel separation plate by such a light detection method, the scattered light from the agarose gel is large and the measurement cannot be performed with high sensitivity. Here, this problem was also solved by detecting immediately after flowing out from the gel.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram of an embodiment of the present invention. FIG. 2 is a sketch drawing of the gel plate and the buffer solution in which the gel separating part used in one embodiment of the present invention is divided. FIG. 3 is a sketch of a gel plate and a buffer solution in which the width of the measuring section used in one embodiment of the present invention is narrowed. 1 ... Light source, 2 ... Mirror, 3 ... Lens / filter system, 4 ... Photodetector, 5 ... Data processor, 6 ... Irradiation unit, 7 ... Gel plate, 8 ... Partition plate, 9 …… Sample injection well, 10 …… Buffer solution, 11 …… Irradiation light, 12 …… Migration bath, 13, 13 ′ …… Electrode, 14 …… Gel end face, 15 …… Migration path separation plate, 16 …… Upper cover, 17 ... Holding plate with steps.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location 9282-4B C12N 15/00 A

Claims (6)

(57) [Claims] 1. A gel, which is placed in a solution and in which a plurality of migration paths for electrophoretically separating a fluorescently labeled sample are formed, and a gel which is placed on both sides of the gel in the direction in which the sample migrates An electrode for applying a voltage for applying an electrophoretic force, a means for irradiating the sample that migrates in the solution along the end surface of the gel on the migration end side where the sample flows out of the gel, and the light And a photodetector for detecting fluorescence emitted from the fluorescent label in the solution, wherein the photodetector is arranged so as to face the end face of the gel on the migration termination side. apparatus. 2. 5 mm from the end surface of the gel on the end point side of the migration
The DNA detection device according to claim 1, wherein the sample is irradiated with the light at the following distances. 3. A gel which is placed in a solution and which electrophoretically separates a fluorescently labeled sample, and a voltage which is placed on both sides of the gel in the direction in which the sample runs to apply an electrophoretic force to the sample. An electrode to be applied, a means for irradiating the sample that migrates in the solution along the end surface of the gel on the migration end side where the sample flows out from the gel, and fluorescence emitted from the fluorescent label by the light. A light detecting means for detecting in a solution, and the gel dividing member divides the gel into a plurality of pieces in a direction substantially parallel to the direction in which the sample migrates, and The DNA detection device is arranged so as to face the end surface of the gel on the side. 4. A gel, which is placed in a solution and in which a plurality of migration paths for electrophoretically separating a fluorescently labeled sample are formed, and a gel which is placed on both sides of the gel in the direction in which the sample migrates An electrode for applying a voltage for applying an electrophoretic force, a means for irradiating the sample that migrates in the solution along the end surface of the gel on the migration end side where the sample flows out of the gel, and the light A DNA detection device comprising: a light detection unit that detects fluorescence emitted from the fluorescent label in the solution, and a thickness of the gel on the injection end side of the sample and a thickness on the migration end point side are different. 5. A gel which is placed in a solution and in which a plurality of migration paths for electrophoretically separating a fluorescently labeled sample is formed, and a gel which is placed on both sides of the gel in the direction in which the sample migrates An electrode for applying a voltage for applying an electrophoretic force, a means for irradiating the sample that migrates in the solution along the end surface of the gel on the migration end side where the sample flows out of the gel, and the light And a light detecting unit for detecting fluorescence emitted from the fluorescent label in the solution, and the thickness of the gel is gradually reduced along the direction from the injection end side of the sample to the migration end point side. Characteristic DNA
Detection device. 6. A plurality of migration paths through which a fluorescence-labeled sample migrates, and a liquid tank through which the sample flows out from the ends of the migration paths,
Means for irradiating the sample flowing out into the liquid tank with laser light in the vicinity of the end of the migration path, and light for detecting fluorescence emitted from the fluorescent label in the liquid tank by irradiation with the laser light. A DNA detection device comprising a detection means.