JP3024742B2 - Fluorescence detection method in multicolor fluorescence detection type electrophoresis apparatus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting fluorescence in a multicolor fluorescence detection type electrophoresis apparatus, and more particularly, to multicolor labeling of DNA whose base sequence is to be determined using a plurality of phosphors having different emission wavelengths. After electrophoretic separation,
The present invention relates to a method for detecting fluorescence in a multicolor fluorescence detection-type electrophoresis apparatus suitable for determining the base sequence of the DNA by detecting emitted fluorescence.

[0002]

2. Description of the Related Art 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 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. The electrophoresis path is irradiated with a laser, and the emitted fluorescence is received by a detector equipped with four types of band-pass 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.

[0003]

It is important to achieve high sensitivity in the above fluorescence detection method. In a measurement system that uses a rotating filter to sweep the detector with irradiation light,
The ratio α of the measurement time per one measurement point is α = d / 4l, where l is the length of the measurement area and d is the width of the irradiation laser beam. Usually d is 0.2 to 0.3 mm, l ≧ 100 mm
Therefore, α ≦ 10 ⁻³ , and only about 10 ^{−3 of the} amount of fluorescent light when continuous light irradiation and light reception are performed is obtained, and there is a problem that high sensitivity cannot be obtained. On the other hand, laser light is incident from the side of the gel plate, each measurement point is continuously irradiated, and the obtained fluorescent image is spectrally separated by a prism and detected by a two-dimensional detector. sell. 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 method that solves this difficulty.

[0004]

Means for Solving the Problems As a result of research, the present inventors have firstly divided a fluorescent image obtained by irradiating a laser onto a gel electrophoresis separation plate into a plurality of virtual images by an image dividing means, The above object is successfully achieved by forming the images on a detector through a process of wavelength-selecting the light of the divided individual images by a band-pass filter and performing required separation and detection. The inventor has found that the present invention is to be carried out, and based on this new finding, conducted further research, and completed the present invention.

Accordingly, the present invention provides a fluorescence detection type electrophoresis apparatus for detecting a sample labeled with different fluorophores by electrophoretically separating the sample with a gel electrophoresis separation section having a plurality of migration paths and then irradiating the sample with a laser beam. In the detection method,
A laser beam is applied to a predetermined position from the migration start point of a plurality of migration paths, and an emission line image (fluorescence image) is placed on the optical path of the laser light.
Forming a plurality of virtual images that are displaced from each other by image-dividing the emission line image, and spatially separating and detecting a plurality of virtual images of the emission line image after each wavelength selection. .

[0006] The multicolor fluorescence detection type electrophoresis apparatus used in the method of the present invention comprises, for example, an image splitting means for splitting a linear emission line image obtained by irradiating a required portion of an electrophoresis separation plate with a laser; Each of the images divided by the image dividing means has a different number of band-pass filters having the same transmission wavelength band as the number of the divided images, divided by the image dividing means, and the individual bands The pass filters are configured to form individual images, each composed of light of a different transmission wavelength, on the fluorescence detector.

[0007] More specifically, the laser irradiation on a required portion of the gel electrophoresis separation plate is performed by a laser irradiation in a direction penetrating from a side surface of the gel electrophoresis separation plate in parallel with a plane of the gel electrophoresis separation plate. Which is a fluorescence image that emits a linear fluorescent image obtained by laser irradiation along the path of laser irradiation in the gel plate.

[0008] 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 constituted by a lens provided in a path leading to an image forming site on the fluorescence detector and having at least a plurality of lenses having different optical axes or a lens obtained by dividing one lens and shifting the optical axis of each fragment. In order to further assist the image splitting function of the image splitting means, the fluorescence emitted from the laser irradiating section of the gel electrophoresis separation plate is image-divided in a path leading to an image formation site on a fluorescence detector. For example, a prism or a mirror may be provided.

The plurality of lenses having different optical axes can be configured by arranging two or four rectangular condensing lenses such that their long sides are horizontal and they are vertically stacked. 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 and condenser lens, and each fragment is apical angled to the center of the lens. The optical axis can be shifted so as to be inclined from the plane by 1 to 2 ° so as to form the surface of the polyhedron.

A specific configuration of a prism or a mirror provided to assist the image dividing function of the image dividing means is such that two mirrors of 10 mm × 100 mm are joined at long sides and deviated by 1 ° from the plane. 5 to 1 from the light irradiation part
A configuration in which a prism having a vertex angle of 150 ° is installed at a position rearward by 0 mm and a vertex is placed at a position 20 mm in front of the irradiation unit so that the vertex is parallel to the irradiation unit, or the like can be given.

When the image dividing means comprises 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 be respectively detected by the fluorescence detectors. It is provided correspondingly in the path leading to the upper imaging site. Preferably, the plurality of reflection mirrors are arranged on an elliptical orbit with the laser irradiation part of the electrophoresis separation plate as one focal point. Specifically, the plurality of reflection mirrors can be configured such that the long sides of four mirrors of 10 mm × 100 mm are combined, and the four mirrors are bent and arranged so as to be in contact with the ellipse.

[0012] A multicolor-labeled sample is used as a sample for separation and detection on the gel electrophoresis separation plate. Fluorescent dyes used for multicolor labeling in this case include FITC (fluorescein isothiocyanate; emission wavelength 515 nm) and NBD-F (4-fluoro-7 nitrobe).
nzofurazan; emission wavelength 540 nm), TRITC (tetra
Methyl rhodamine isothiocyanate; emission wavelength 573) and Texas Red (emission wavelength 610 nm) can be used. As a bandpass filter corresponding to the multicolor marker, a multilayer dielectric filter or the like having a transmission band whose center wavelength matches the above-mentioned emission wavelength and a transmission band width of 20 to 40 nm is used. Usually, a two-dimensional fluorescence detector is used as the fluorescence detector.

According to the fluorescence detection type electrophoresis apparatus of the present invention,
As the target sample for separation and detection, D
Examples include NA and RNA, but proteins and the like can also be used as target samples. 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. Is detected.

Therefore, according to the present invention, fluorescences having different emission wavelengths can be separated and detected with high accuracy without time-division detection, that is, without detecting them temporally. Therefore, the apparatus of the present invention can be suitably used for determining the base sequence of a multicolor fluorescently labeled DNA 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.

[0015]

FIG. 1 is a block diagram showing an embodiment of the present invention.
This will be described with reference to FIG. Two glass plates 1 at 0.3 mm intervals
6% polyacrylamide gel plate 2 sandwiched between (300 mm × 200 mm × 5 mm) glass (or quartz)
Argon laser (400 nm, 10 mw) light 4 is guided from a laser light source 3 and made incident from the side surface of the gel plate 2 sandwiched between the plates 1. 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 downward. 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 a difference in migration path, the 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-
Although F, TRITC and Texas Red are used, 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 with the electrophoresis plate interposed therebetween, and the fluorescent image is reflected from the detector side. Make the four virtual images 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. I have. The four virtual images are imaged by the lens 7 on the two-dimensional detector 9 as four lines. 515 corresponding to four kinds of phosphors are provided in front of each image forming position.
A band-pass filter 8 having transmission bands at nm, 573 nm, 540 nm, and 610 nm is provided to 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 is a monitor 1
Four lines 14 as shown in FIG. Each is a signal from a fluorescent substance having a different wavelength, and thus a DNA fragment having 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. 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 irradiation unit from directly entering the light receiving unit, a 1 mm wide strip-shaped reflection mirror 15 is attached to the glass plate by vapor deposition on the glass plate in contact with the gel. . 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. 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. Two glass plates 1 (30
A gel plate 2 of 6% polyacrylamide sandwiched between 0 mm × 200 mm × 5 mm) was placed on the side of the gel plate 2 sandwiched between glass (or quartz) plates 1 using an argon laser (400 mm).
nm 10 mw) Light 4 is guided from the laser light source 3 and made 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 material is separated on a different electrophoresis path for each sample according to the length. 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. In this embodiment the quadruple image a fluorescent image using a birefringent prism 16 and split lens _71, each of the image passes through the band-pass filter _81, 82 corresponding to the four kinds of labeling fluorescent It is devised to form an image on the two-dimensional detector 9. The divided lens ₇₁ is a focal length 30m
m, two 30 mm × 50 mm rectangular lenses. Each of the bandpass filters 8 ₁ and 8 ₂ includes a dielectric multilayer filter having two band-shaped different transmission wavelength bands. Center transmission wavelength is 515n
m, 540 nm set and 575 nm, 610 nm
, But this combination may vary.

The image is doubled by the birefringent prism and further divided by the split lens whose lens axis is slightly shifted.
One image at a time. 4 instead of using a birefringent prism
Lenses that are divided and whose axes are slightly shifted from each other may be used. In still another embodiment, it can also be used four lens 7 ₂ as shown in FIG. The four lenses 7 ₂
Is a structure in which four rectangular lenses are joined at the long side so that four optical lenses are different from each other.

The band-pass filter 8 ₃ in this embodiment is composed of 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. For image division in the embodiment shown in FIG. 3 or FIG. 4, a reflecting mirror or a prism may be used together with a birefringent prism or a split lens.

[0023]

According to the present invention, first, a plurality of fluorescent images are formed using the image dividing means, and each image is formed on a light receiving device through a filter which identifies and transmits light having different wavelengths. Therefore, compared to the conventional method of separation and detection using only prism 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 showing an example of an apparatus used in the method of the present invention.

FIG. 2 is a sectional view of an optical system used in the apparatus of FIG.

FIG. 3 is a schematic view showing another example of the apparatus used in the method of the present invention.

FIG. 4 is a schematic view showing another example of the apparatus used in the method of the present invention.

[Explanation of symbols]

1: glass (quartz) plate, 2: gel plate, 3: laser, 4
... Laser light, 5 ... Reflection mirror, 6 ... Excitation light cut filter, 7 ... Lens, 71,72 ... Division lens, 8 ... Band pass filter, 81 ... Upper filter, 82 ... Lower filter, 83 ... Mosaic filter, 9 ... Two-dimensional detector, 10 ... control device, 11 ... data processing device, 12
... display device, 13 ... monitor, 14 ... observed line image, 15 ... reflection mirror, 16 ... birefringent prism

Continuation of front page (56) References JP-A-61-292027 (JP, A) JP-A-63-231247 (JP, A) JP-A-55-6293 (JP, A) JP-A-63-209288 (JP) , A) (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 21/00-21/65

Claims (5)

(57) [Claims] 1. A multicolor fluorescence detector for separating a sample labeled with a different fluorescent substance by electrophoresis in an electrophoretic separation section having a plurality of migration paths and then irradiating a laser beam to detect the fluorescent light emitted from the fluorescent substance. In the fluorescence detection method in a type electrophoresis apparatus, a laser beam is applied to the plurality of migration paths to form a fluorescence image on the optical path of the laser light, and a portion of the electrophoresis unit to which the laser light is applied is formed. a plurality of reflecting mirrors disposed on the elliptical peripheral line to one focus, dividing the fluorescence-ray image into a plurality of images, the plurality of reflection
A method for detecting fluorescence in a multicolor fluorescence detection-type electrophoresis apparatus, comprising: passing light reflected by a mirror through bandpass filters having transmission bands different from each other. 2. A multicolor fluorescence detector for separating a sample labeled with different phosphors by electrophoresis in an electrophoresis separation section having a plurality of migration paths and then irradiating a laser beam to detect fluorescence emitted from the phosphors. In the fluorescence detection method in the electrophoretic device, a birefringent prism is formed by irradiating the plurality of migration paths with a laser beam to form a fluorescence image on the optical path of the laser beam and facing the fluorescence image. The fluorescent ray image is divided into a plurality of images by a lens which is divided into fragments having different optical axes, and the image is passed through the birefringent prism and the lens.
A method for detecting fluorescence in a multicolor fluorescence detection-type electrophoresis apparatus, characterized in that the transmitted light is passed through bandpass filters having different transmission bands. 3. A multicolor fluorescence detector which separates samples labeled with different fluorescent substances by electrophoretic separation in an electrophoretic separation section having a plurality of migration paths, and irradiates a laser beam to detect fluorescent light emitted from the fluorescent substances. In the fluorescence detection method in the electrophoresis apparatus, the plurality of migration paths are irradiated with laser light to form a fluorescence image on the optical path of the laser light .
Light et light has passed through the band-pass filter having different transmission bands, different by fragments divided lens having an optical axis into a plurality of image in the vertical direction parallel to a plurality of linear image with each other A method for detecting fluorescence in a multicolor fluorescence detection type electrophoresis apparatus, characterized by forming: 4. A multicolor fluorescence detector for separating a sample labeled with a different fluorescent substance by electrophoresis in an electrophoretic separation section having a plurality of migration paths and then irradiating a laser beam to detect fluorescent light emitted from the fluorescent substance. In the fluorescence detection method in the type electrophoresis apparatus, the plurality of migration paths are simultaneously irradiated with laser light to form a fluorescence image on the optical path of the laser light, and the image is divided.
Means to divide the fluorescent image in the vertical direction to form a plurality of linear images parallel to each other, and pass through the image dividing means.
The center wavelength of the transmission band is equal to the emission wavelength of the phosphor.
The width of the transmission band is in the range of 20 nm to 40 nm.
Ri is passed through a bandpass filter with a different transmission bands, a plurality of parallel lines corresponding to the emission wavelength of the fluorescent
A method for detecting fluorescence in a multicolor fluorescence detection type electrophoresis apparatus, wherein images are formed at different positions on a photodetector. 5. A multicolor fluorescence detector for separating a sample labeled with a different fluorescent substance by electrophoresis in an electrophoretic separation section having a plurality of migration paths and then irradiating a laser beam to detect fluorescent light emitted from the fluorescent substance. In the fluorescence detection method in a type electrophoresis apparatus, the plurality of migration paths are simultaneously irradiated with laser light to form a fluorescence image on the optical path of the laser light, and light from the fluorescence image is centered on a transmission band. The emission wavelength of the phosphor is
The transmission band width is in the range of 20 nm to 40 nm according to the wavelength.
The light passing through the band-pass filters having different transmission bands in the range is divided in the vertical direction to form a plurality of parallel linear images, and the light corresponds to the emission wavelength of the fluorescence.
A method for detecting fluorescence in a multicolor fluorescence detection type electrophoresis apparatus, wherein a plurality of parallel line images are formed at different positions on a photodetector.