JP2809422B2 - Multicolor fluorescence detection type electrophoresis device - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a multicolor fluorescence detection type electrophoresis apparatus, and more particularly, to a DNA for which a base sequence is to be determined using a plurality of phosphors having different emission wavelengths. The present invention relates to a multicolor fluorescence detection-type electrophoresis apparatus suitable for determining the base sequence of the DNA by detecting fluorescence emitted after multicolor labeling and electrophoretic separation.

[Conventional technology]

Conventionally, DNA sequencing has been performed by autoradiography using radioisotope labeling. However, recently, a technique of automatically detecting a DNA fragment optically using a fluorescent label and automatically determining a base sequence has become widespread. In this method, four types of DNA fragment groups having different terminal base types are labeled with fluorescent materials having different emission wavelengths, and the DNA fragments are separated by gel electrophoresis. Irradiate with laser on electrophoresis path,
The emitted fluorescence is received by a detector equipped with four types of bandpass filters that maximize the transmission wavelength of each emission wavelength. As a detector, a method of moving a photomultiplier tube having four types of bandpass filters on a rotating plate in synchronization with a laser beam to be scanned is used. Further, it has been proposed to irradiate the electrophoresis plate with a laser in a line shape, to separate a linear fluorescence image with a prism, and to detect the fluorescence image using a high-sensitivity two-dimensional detector.

[Problems to be solved by the invention]

It is important to achieve high sensitivity in the above fluorescence detector. In a measurement system that sweeps the detector with irradiation light using a rotating filter, the ratio α of the measurement time per one measurement point of the gel is as follows, where l is the length of the measurement area and d is the width of the irradiation laser beam. Becomes Usually d is 0.2-0.3mm, l100mm, so α10
^-3 , which means that only about 10 ^{-3 of the} amount of fluorescence received when continuous light irradiation and light reception were performed was obtained, and there was a problem that high sensitivity could not be obtained. On the other hand, laser light is incident from the side of the gel plate, each measurement point is continuously irradiated, the obtained fluorescent image is spectrally separated by a prism and detected by a two-dimensional detector, the amount of received light is large, and this difficulty can be overcome. . However, the spectral accuracy by the prism is low, and there is a problem in highly accurate base identification.

An object of the present invention is to provide a fluorescence detection device that solves this difficulty.

[Means for solving the problem]

As a result of the study, the present inventors first divided a fluorescent image obtained by laser irradiation on the gel electrophoresis separation plate into a plurality of virtual images by image dividing means, and then illuminated the light of each of the divided images. Through a process of wavelength selection by a band-pass filter, these images were formed on a detector to perform the required separation and detection, and found that the above object was successfully achieved. Based on further studies, the present invention has been completed.

Therefore, the multicolor fluorescence detection type electrophoresis apparatus of the present invention includes a gel electrophoresis separation unit having a plurality of migration paths on which samples labeled with different fluorophores migrate, and a predetermined electrophoretic separation from a migration start point of the plurality of migration paths. A plurality of light irradiation means for irradiating the position with excitation light to excite the phosphor to form a fluorescent image, a photodetector, an image dividing means and an image forming means, wherein the fluorescent image is displaced from each other; An optical system that divides the image into virtual images and then forms an image at a plurality of imaging positions separated from each other on the photodetector, and each light from the light irradiation site of the gel electrophoresis separation unit to the plurality of imaging positions of the photodetector A plurality of wavelength selectors each having a different transmission wavelength band disposed on a road, wherein the photodetector has a plurality of different wavelength bands that pass through each of the plurality of wavelength selectors and are imaged at an imaging position. Is detected.

The wavelength selection means may be bandpass filters having different transmission wavelength bands.

As a practical configuration of the apparatus of the present invention, a direction in which laser irradiation to a required portion of the gel electrophoresis separation plate penetrates from a side surface of the gel electrophoresis separation plate in parallel with a plane of the gel electrophoresis separation plate. Is a fluorescent image generated by emitting a linear fluorescent image obtained by laser irradiation along a path of laser irradiation in the gel plate.

The image dividing means is constituted by a plurality of reflection mirrors arranged in parallel with the laser irradiation part of the gel electrophoresis separation plate as an axis, or the fluorescence emitted from the laser irradiation part of the gel electrophoresis separation plate is detected by fluorescence. At least a plurality of lenses with different optical axes, or a lens in which one lens is divided and the optical axis of each fragment is shifted, provided in a passage leading to an image formation site on the container In order to further assist the image dividing function of the image dividing means, the fluorescence emitted from the laser irradiating portion of the gel electrophoresis separation plate passes through the passage to the image formation site on the fluorescence detector, for image division. Prisms or mirrors can also be provided.

The plurality of lenses having different optical axes may be configured by arranging two or four rectangular condensing lenses so that the long sides are horizontal and vertically.

The lens in which the one lens is divided and the optical axis of each fragment is shifted is divided into 7 or 4 fragments by a line passing through the center of the circular, condenser lens, and each fragment is apical angled at the center of the lens. The optical axis can be shifted so as to incline from the plane of 1 to 2 ° so as to form the surface of the polyhedron.

As a specific configuration of the prism or mirror attached to assist the image dividing function of the image dividing means, 10 mm
Two x100mm mirrors are joined at the long side, and set at a position 5 to 10mm behind the light irradiating part with a deviation of 1mm inside and outside from the plane, and a prism with a vertex angle of 150mm is placed 20mm in front of the irradiating part. A configuration in which the apex is set so as to be parallel to the irradiation unit can be given.

When the image dividing means includes a plurality of reflection mirrors, the same number of band-pass filters having different transmission wavelength bands as the number of the reflection mirrors cause the fluorescence reflected by the reflection mirrors to form light on the fluorescence detector. It is provided correspondingly in the passage leading to the image site.

Preferably, the plurality of reflection mirrors are arranged on a circular orbit with the laser irradiation part of the electrophoresis separation plate as one focal point.

The plurality of reflection mirrors are, specifically, 10 mm × 100 mm
The four mirrors can be configured such that the long sides of the four mirrors are combined and the four mirrors are folded and arranged so as to be in contact with the ellipse.

Further, when the apparatus of the present invention is a multicolor fluorescence detection-type electrophoresis apparatus used for separation and detection of a multicolor-labeled sample, the multicolor-labeled sample is subjected to multicolor labeling as a separation detection sample for electrophoresis of a gel electrophoresis separation plate. A sample is used. The fluorescent dye used for multicolor labeling in that case includes FITC (fluorescein isothiocyanate; emission wavelength 515n).
m), NBD-F (4-fluoro-7 nitrobenzofurazan; emission wavelength 540 nm), TRITC (tetramethy1 rhodamine isothiocy
anate; emission wavelength 573) and Texas Red (emission wavelength 610n)
m) can be used.

For a bandpass filter corresponding to a multicolor marker, the center wavelength of the transmission band coincides with the above emission wavelength, and the width of the transmission band is
A multilayer dielectric filter of 20 to 40 nm or the like is used.

Usually, a two-dimensional fluorescence detector is used as the fluorescence detector.

As a sample to be separated and detected by the fluorescence detection type electrophoresis apparatus of the present invention, DNA or RNA whose base sequence is to be determined
However, proteins and the like can also be used as the target sample.

(Operation)

According to the present invention, a fluorescent image obtained by irradiating a laser on a gel electrophoresis separation plate firstly includes a reflecting mirror or a plurality of lenses having different optical axes, or split lenses having optical axes shifted from each other. First, the image is divided into a plurality of virtual images by the image dividing means, and then the light of each of the divided images is subjected to a wavelength dispersion process by a band-pass filter, and these images are formed on a detector. In this case, separation and detection of places are performed.

Therefore, in the present invention, fluorescences having different emission wavelengths can be separated and detected with high accuracy at the same time without detecting them in a time-division manner, that is, time-sequentially.

Therefore, the device of the present invention is a multicolor fluorescently labeled DN
It can be suitably used for determining the nucleotide sequence of the A fragment.

In addition, since the irradiation section is continuously irradiated from the side and the entire irradiation area is simultaneously observed with the two-dimensional detector, the amount of received light is large and high sensitivity can be obtained.

〔Example〕

Hereinafter, an embodiment of the present invention will be described with reference to FIG.
A 6% polyacrylamide gel plate 2 sandwiched between two 0.3 mm glass plates 1 (300 mm × 200 mm × 5 mm) is an argon laser (from the side of the gel plate 2 sandwiched between glass (or quartz) plates 1). 400nm 10mw) Light 4 to laser light source 3
And then let it enter. The irradiation part is represented by a broken line in the sectional view. DN labeled with four-color fluorescent substance in four kinds of terminal bases
Sample A is injected at the top of the gel and migrates down. Although a plurality of migration paths can be secured, the laser beam 4 irradiates all the migration paths. Fluorescence is emitted along the irradiation part. In the case of one of the conventional methods, in which the label is monochromatic and the terminal base species is identified by the difference in the migration path, this fluorescent image is put on a line sensor or a two-dimensional detector 9 by a lens 7 with a filter 6. An image is formed and the time change of the fluorescence intensity is observed.
As the four-color phosphor, FITC, NBD-F, TRITC and Texas Red described above were used, but a metal complex phosphor can also be used. In this embodiment using four-color phosphors, four rectangular mirrors of 1 cm in length and 10 cm in width are installed on the opposite side of the detector across the electrophoresis plate, reflect the fluorescence image, and four mirrors from the detector side. Make the virtual image visible. The mirror 5 is arranged on the circumference of an ellipse having a major axis of 20 cm with the pupil position of the lens 7 and the laser irradiation position of the gel as focal points so that the distances to the four virtual images are equal when viewed from the detector side. The four virtual images are imaged by the lens 7 on the two-dimensional detector 9 as four lines. Bandpass filters 8 having transmission bands at 515 nm, 573 nm, 540 nm, and 610 nm corresponding to four kinds of phosphors are provided at all of the image forming positions, and can perform wavelength selection. The band-pass filter need only be between the mirror and the detector, not just before the two-dimensional detector. The image observed by the two-dimensional detector has four lines 14 as shown on the monitor 13.
It is. Each is a signal from a phosphor of a different wavelength, and thus a DNA fragment of a different terminal base type. The horizontal axis corresponds to the horizontal direction of the electrophoresis plate, and the signal at the same position on the abscissa of the four line images is information from the sample included in the same migration path, and the terminal base type is determined by comparing this information. it can. FIG. 2 is a detailed view of the optical system. The fluorescent light reflected by the four mirrors forms a virtual image on the circumference centered on the pupil position of the light receiving lens. In order to increase the amount of received light and at the same time prevent the fluorescence from the irradiated part from directly entering the light-receiving part, a 1 mm wide strip-shaped reflective mirror 1 on the glass plate on the side in contact with the gel
5 is attached by vapor deposition on a glass plate. The width of the mirror is large enough to reflect all the light that can enter the receiver.

The mirror can be installed between the detector and the migration plate. Further, the optical path may be adjusted in combination with a mirror with a prism.

Hereinafter, another embodiment of the present invention will be described with reference to FIG.
A 6% polyacrylamide gel plate 2 sandwiched between two 0.3 mm glass plates 1 (300 mm × 200 mm × 5 mm) is an argon laser (from the side of the gel plate 2 sandwiched between glass (or quartz) plates 1). 400 nm 10 nm) Light 4 is incident. The irradiation part is represented by a broken line in the sectional view. A DNA sample labeled with a four-color fluorescent substance according to four terminal base types is injected into the upper part of the gel and migrates downward. Usually, 20 to 30 migration paths are secured on one migration plate. The laser beam 4 irradiates all the migration paths simultaneously. Fluorescence is emitted along the irradiation part.

The fragment group labeled with the four-color fluorescent substance is separated according to the length on a different electrophoresis path for each sample. When passing through the laser irradiation part, it emits fluorescence of various wavelengths depending on the terminal base type. These fluorescences are observed as line images along the irradiation area. When the fluorescence is separated by a prism or the like, the wavelength dispersion is small, and the respective signals cannot be separated sufficiently. Bandpass filters are excellent for wavelength separation of each fluorescence. A fluorescent image using a plurality refraction prism 16 and split lens ₇₁ in the present embodiment the four image, band-pass filter _81, each image is corresponding to four labeling fluorescent, two passes through the 8 ₂ It is devised to form an image on the dimensional detector 9. The divided lens ₇₁ is made up of focal length 30 mm, 2 sheets of 30 mm × 50 mm rectangular lenses. The band pass filters 8 ₁ and 8 ₂
Are each provided with a dielectric multilayer filter having two band-shaped different transmission wavelength bands. The center transmission wavelength is
A set of 515 nm, 540 nm and a set of 575 nm, 610 nm, but this combination may vary.

The images are doubled by the birefringent prism, and further split into two images by a split lens whose lens axis is slightly shifted. Instead of using a birefringent prism, it is divided into four,
Lenses whose axes are slightly shifted from each other may be used.

In yet another embodiment, it can also be used four lens 7 ₂ as shown in Figure 4. The four lens 7 _2, the four lenses rectangular so as to bond with the long side portion, the optical axis is configured to differ from one another in an ordered four.

Band-pass filter 8 ₃ in this embodiment is constituted by a dielectric multilayer film made of the same material as the filter described above. Then, the band-pass filter 8 ₃ can also be mounted in front of each lens. The image monitored on the two-dimensional detector looks like 14. The portion corresponding to each observation line is read out, and the mutual intensity is corrected by correction calculation, thereby obtaining the intensity change of each fragment group.

In the case of the embodiment shown in FIG. 3 or FIG. 4, a reflecting mirror or a prism may be used in combination with a birefringent prism or a split lens.

〔The invention's effect〕

According to the present invention, first, a plurality of fluorescent images are formed using the image dividing means, and then each image is formed on a light receiving device through a filter that identifies and transmits light having different wavelengths. Compared to the method of separation and detection using only spectroscopy, signals from multicolor-labeled DNA fragments and the like can be distinguished and measured simultaneously with high accuracy, and separation and detection or base sequence determination with high accuracy can be performed.

[Brief description of the drawings]

FIG. 1 is a schematic view of an apparatus configuration of one embodiment of the present invention, FIG. 2 is a cross-sectional view of an optical system used in the apparatus of Embodiment 1, and FIG. 3 is a schematic view of an apparatus configuration of another embodiment of the present invention. FIG. 4 is a schematic view of a device configuration of still another embodiment. 1 ... Glass (quartz), 2 ... Gel plate, 3 ... Laser, 4 ... Laser light, 5 ... Reflection mirror, 6 ... Excitation light cut filter, 7 ... Lens, 7 ₁ , 7 ₂ ... … Division lens, 8… Band pass filter, 8 ₁ …… Upper filter, 8 ₂ …… Lower filter, 8 ₃ …… Mosaic filter, 9 …… 2 dimensional detector, 10 …… Control device, 11 …… Data Processing unit, 12 Display unit, 13 Monitor, 14 Unit
Observed line image, 15 ... reflection mirror, 16 ... birefringent prism.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) G01N 21/62-21/74 G01N 21/00-21/01 G01N 21/17-21/61 G02B 5 / 04 G02B 3/10

Claims (10)

(57) [Claims] 1. A plurality of reflection means which are arranged in parallel with a laser irradiation part where a plurality of migration paths on which a sample migrates are irradiated with laser light, and divide a fluorescence image formed on the laser irradiation part into a plurality of images. A mirror, a photodetector that separates and detects the plurality of images, and a wavelength selection unit that is respectively disposed in an optical path of each fluorescent ray image that is reflected by the reflection mirror and reaches the photodetector. The plurality of reflection mirrors are arranged on an elliptical circumference having the laser irradiation section as one focal point, and the photodetector detects a plurality of fluorescent ray images whose wavelengths are selected by the wavelength selection means. A multicolor fluorescence detection type electrophoresis apparatus, characterized in that: 2. A light irradiating means for irradiating a plurality of migration paths on which a sample labeled with a different fluorescent substance migrates with laser light to excite the fluorescent substance to form a fluorescent ray image, a photodetector, An optical system including an image splitting unit and an image forming unit, which divides the fluorescence line image into an image and forms an image at a plurality of image forming positions separated from each other on the photodetector; and irradiates the plurality of migration paths with the laser beam. A plurality of wavelength selectors each having a different transmission wavelength band disposed on each optical path from a light irradiation site to be formed to a plurality of image forming positions of the photodetector; It has a plurality of reflection mirrors arranged in parallel with the part and arranged on the circumference of an ellipse having the laser irradiation unit as one focal point, and the photodetector has a plurality of wavelength selection units. Wavelength band that passes through and is imaged at the imaging position Multicolor fluorescence detection type electrophoresis apparatus characterized by detecting a plurality of different fluorescent ray images. 3. A light irradiation means for irradiating a migration path on which a plurality of samples labeled with different fluorescent substances are respectively migrated with laser light to excite the fluorescent substance to form a fluorescent ray image, and the plurality of migration paths. Image splitting means comprising a birefringent prism and a lens split into fragments having different optical axes, which are arranged to face the laser irradiation unit where the laser is irradiated,
Photodetector, comprising a plurality of wavelength selection means having different transmission wavelength bands arranged on the optical path from the laser irradiation unit to a plurality of imaging positions of the photodetector, the photodetector, A multicolor fluorescence detection type electrophoresis apparatus, wherein a plurality of the fluorescence ray images having different wavelength bands formed at the image formation positions are detected. 4. An electrophoresis section for separating samples labeled with a plurality of types of phosphors having different emission wavelengths, and irradiating a laser beam while electrophoresing the separated samples to excite the phosphors. A light irradiation unit that generates a fluorescent line image from the electrophoresis unit, a dividing unit that vertically divides the phosphor line image to form a plurality of parallel line images, and each of the plurality of line images. A plurality of band-pass filters for transmitting light of correspondingly different wavelength bands, and a plurality of line images of different wavelength bands passing through the plurality of band-pass filters are converted into parallel line images to form one two-dimensional light detection. A multicolor fluorescence detection type electrophoresis apparatus characterized in that the electrophoresis apparatus separates and forms an image at different positions of a vessel and detects it. 5. A multicolor fluorescence detection type electrophoresis apparatus according to claim 4, wherein said image dividing means has a plurality of rectangular condensing lenses having different optical axes arranged vertically with a long side being horizontal. A multicolor fluorescence detection type electrophoresis apparatus, comprising: 6. An electrophoresis section for separating samples labeled with a plurality of types of phosphors having different emission wavelengths, and irradiating the separated sample with a laser beam while exciting the sample to excite the phosphor. A light irradiating unit that generates a fluorescent line image from the electrophoretic unit; an image dividing unit that vertically divides the fluorescent line image to form a plurality of parallel line images; A plurality of bandpass filters that pass light of different wavelength bands corresponding to each of the plurality of line images to be processed, and a plurality of line images having different wavelength bands that have passed through the plurality of bandpass filters are parallelized. 1 as a line image
A multicolor fluorescence detection type electrophoresis apparatus characterized in that two two-dimensional photodetectors are separated at different positions to form an image and detected. 7. A multicolor fluorescence detection type electrophoresis apparatus according to claim 6, wherein said image splitting means has a plurality of rectangular condensing lenses having different optical axes arranged vertically with a long side being horizontal. A multicolor fluorescence detection type electrophoresis apparatus, comprising: 8. A laser irradiation unit that irradiates a plurality of migration paths on which a sample labeled with a plurality of types of fluorescent substances having different emission wavelengths migrates with laser light to excite the fluorescent substances and is irradiated with the laser light. A light irradiating means for forming a fluorescence image, a plurality of reflection mirrors arranged in parallel with the laser irradiation unit to divide the fluorescence image into a plurality of line images, and one two-dimensional light from the plurality of reflection mirrors Wavelength selecting means arranged in each optical path of the plurality of line images reaching the detector, wherein the two-dimensional photodetector includes a plurality of lines having different wavelength bands selected by the wavelength selecting means. A multicolor fluorescence detection type electrophoresis apparatus, wherein an image is separated and formed as a parallel line image at different positions of the two-dimensional photodetector and detected. 9. A laser irradiation section which irradiates a plurality of migration paths on which a sample labeled with a plurality of kinds of phosphors having different emission wavelengths migrates with laser light to excite the phosphor and is irradiated with the laser light. A light irradiating means for forming a fluorescent ray image, a plurality of reflecting mirrors arranged in parallel to the laser irradiating part and reflecting the fluorescent ray image, a wavelength selecting means and one two-dimensional photodetector An optical system, wherein the fluorescence ray images reflected from the plurality of reflection mirrors and emitted from apparently different positions are formed and detected at different positions of the two-dimensional photodetector as a plurality of line images having different wavelength bands. And a multicolor fluorescence detection type electrophoresis apparatus. 10. The multicolor fluorescence detection type electrophoresis apparatus according to claim 9, wherein the plurality of reflection mirrors are arranged on an elliptical line having the laser irradiation section as one focal point. A multicolor fluorescence detection type electrophoresis apparatus characterized by the following.