JPH08261935A - Fluorescence detecting method for fluorescence detection type electrophoretic device - Google Patents

Abstract

PURPOSE: To provide a fluorescence detecting method capable of concurrently distinguishing the signals from DNA fragments marked in many colors with high accuracy and measuring them. CONSTITUTION: A fluorescent image obtained by the laser radiation to a gel electrophoresis separator 2 is image-split into multiple virtual images by prisms 4, 5, the light of the individual split images is wavelength-dispersed by band-pass filters 7, 8, then the images are formed on a detector 9 arid separated and detected as required.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence detection method in a fluorescence detection type electrophoretic device, and more specifically, it uses a plurality of fluorophores having different emission wavelengths to label a DNA whose base sequence is to be determined, and then performs electrophoresis The present invention relates to a fluorescence detection method in a fluorescence detection-type electrophoresis apparatus suitable for determining the base sequence of the DNA by detecting the fluorescence emitted after separation.

[0002]

2. Description of the Related Art DNA fragments having different terminal base species are labeled with fluorophores having different emission wavelengths, and a certain position of the migration path is irradiated with light while being subjected to gel electrophoresis separation. A technique for determining the terminal base species of a DNA fragment passing through the irradiation part and determining the sequence has been developed.
To identify and detect the fluorescence wavelength, use a rotary filter equipped with four types of bandpass filters in front of the photomultiplier tube, or use two photomultiplier tubes equipped with filters with different transmission wavelength bands. are doing. In either case, the detector is swept across multiple migration paths and multiple samples D
The base sequence of NA has been determined. On the other hand, a method in which laser light is incident from the side of the gel plate, each measurement point is continuously irradiated, and the resulting fluorescence image is dispersed by a prism and detected by a two-dimensional detector is also available. Proposed.

However, in the measurement system in which the above-mentioned detector is swept, the ratio α of the measurement time per one measurement point of the gel is α
Is the length of the measurement area is l and the width of the irradiation laser beam is d
Then, α = d / 4l. Usually d is 0.2 to 0.3 mm, l ≧ 100 mm
Therefore, α ≦ 10 ⁻³ , and only a fluorescent light receiving amount of about 10 ⁻³ can be obtained when continuous light irradiation and light reception are performed, and high sensitivity cannot be obtained. On the other hand, disperse with the above prism,
In the method of detecting by the two-dimensional detector, the received light amount is large and this difficulty can be overcome. However, the spectral accuracy of the prism is low, and there is a difficulty in highly accurate base identification.

[0004]

As described above, in the prior art, no consideration was given to enhancing the sensitivity, or no consideration was given to wavelength separation with high precision, and it was not possible to determine the base sequence with high precision. An object of the present invention is to solve the above problems and provide a method capable of highly accurate and highly sensitive DNA separation detection.

[0005]

Means for Solving the Problems As a result of research by the present inventors, the fluorescence image obtained by irradiating the gel electrophoresis separation plate with a laser is first divided into a plurality of virtual images by an image dividing means such as a prism, and then, By performing a process of wavelength-dispersing the light of each of the image-divided individual images by a bandpass filter, these images are formed on a detector to perform required separation detection, It has been found that the object can be achieved satisfactorily, and further research has been conducted based on this new finding to complete the present invention.

Therefore, the present invention provides a fluorescence detection method in a fluorescence detection type electrophoretic apparatus in which samples labeled with different fluorescent substances are electrophoretically separated in a plurality of migration paths and then detected by irradiating laser light. A step of irradiating a plurality of migration paths with laser light at a predetermined position from the migration start point to excite the phosphor to form a light emission line image along a path irradiated with the laser light; A step of dividing the image into a plurality of images, a step of selecting a wavelength in each optical path of the light of the plurality of divided images, and a step of forming the plurality of divided images on a photodetector. It is characterized in that fluorescence from a fluorophore that labels the separated sample is detected.

The emission line image is formed along the path irradiated with the laser by irradiating the laser light so as to penetrate through the plurality of migration paths from the direction intersecting with the migration path. The fluorescence detection type electrophoretic device used in the method of the present invention is a linear light source obtained by irradiating at least a laser light source, a gel electrophoresis separation plate, a fluorescence detector, and a laser to a required portion of the gel electrophoresis separation plate. A prism having a plurality of optical planes for dividing the fluorescence image, and an optical system for individually forming a plurality of images divided by the plurality of optical planes of the prism on the fluorescence detector. Is preferred.

As a practical configuration of the apparatus, laser irradiation to a required portion of the gel electrophoretic separation plate penetrates from a side surface of the gel electrophoretic separation plate in parallel with a plane of the gel electrophoretic separation plate. Direction laser irradiation, and a linear fluorescence image obtained by laser irradiation is emitted along the laser irradiation path in the gel plate.

An optical system is provided with a bandpass filter having different transmission wavelength bands respectively installed correspondingly in the light paths of the divided images emitted from a plurality of optical planes of the prism. It is said that

As a concrete shape of the prism, a polyhedron symmetrical to the cross section including the ridge of the apex angle of the central portion as shown in the drawings for explaining the embodiments, or a symmetrical cross section to the central portion like a trapezoid. There are various polyhedra. The material of the prism must be a material having a high refractive index, particularly when the device of the present invention is used as a multicolor fluorescence detection type electrophoretic device. L
Examples thereof include aF3, SF3, glass and the like.

Further, when the method of the present invention is applied to a multicolor fluorescence detection type electrophoretic device used for separation and detection of multicolor-labeled samples, many samples are used as separation and detection samples for migrating a gel electrophoresis separation plate. A color labeled sample is used. The fluorescent dye used for multicolor labeling in that case is FITC (fluorescein isothiocyana).
te; emission wavelength 515 nm), NBD-F (4-fluoro-7 ni)
trobenzofurazan; emission wavelength 540nm), TRITC
(Tetramethyl rhodamine isothiocyanate; Emission wavelength 5
73 nm) and Texas Red (emission wavelength 610
nm) or a phosphor containing a metal complex.

As a bandpass filter corresponding to a multicolor mark, a combination of a dielectric vapor deposition multilayer film filter and a colored glass filter is used. Moreover, as a desirable arrangement of the prism, at least one apex angle of the prism is
An emission line image of a linear fluorescence image obtained by irradiating a required portion of the electrophoretic separation plate with a laser and a plane including the center of an image formation site of the fluorescence detector, and an emission line image of the fluorescence image. Be placed in parallel, or
The prism is vertically symmetrical with respect to the plane including the emission line image of the linear fluorescence image obtained by irradiating the desired portion of the gel electrophoresis separation plate with the laser and the center of the image formation site of the fluorescence detector, and The apex angle of the central portion is set to be parallel to the emission line image of the fluorescence image.

A two-dimensional fluorescence detector is usually used as the fluorescence detector. The sample to be separated and detected by the fluorescence detection method of the present invention is DN whose base sequence is to be determined.
Examples of the target sample include A and RNA, but proteins and the like can also be used as the target sample.

Although the method of the present invention is most preferably applied to a multicolor fluorescence detection type electrophoresis apparatus as described above, it can also be used in the case of monochromatic fluorescence detection. Also in this case, using the method of the present invention, the linear fluorescence image is divided into, for example, two images so that the light of the fluorescence peak is measured by the former and the light of a specific low wavelength is measured by the latter. By selecting a combination of bandpass filters, there is a merit that it is possible to correct a measurement error due to a change in measurement conditions such as fluctuation of laser irradiation light and perform accurate separation detection.

According to the present invention, the fluorescence image obtained by irradiating the gel electrophoresis separation plate with the laser is first image-divided into a plurality of virtual images by the prism, and then the light of each image-divided image is divided. These images are formed on a detector through a process such as wavelength dispersion by a bandpass filter, and required separation detection is performed.

More specifically, in the fluorescence detection type electrophoretic device used in the method of the present invention, when the irradiation portion is viewed from the pupil position of the light receiving lens, the apex angle of the central portion of the prism is preferably linear. It is placed so that it overlaps with the light emitting part. Then, the light passing through the upper half part and the lower half part of the prism is imaged on the two-dimensional detector as two images like light emitted from different points, and wavelength dispersion is caused in the vertical direction. The light coming from each prism passes through different filters and reaches the detection part.The signals with similar wavelengths are separated and detected depending on whether they have passed through the upper half or the lower half of the prism. Dispersion can be detected by wavelength dispersion.
According to the present invention, fluorescence having different emission wavelengths can be simultaneously and separately detected with high accuracy by the combination of the prism and the filter without time-division, that is, detection with time division.

Therefore, the method of the present invention can be suitably used for determining the base sequence of a multicolor fluorescently labeled DNA fragment. Further, since the irradiation part is continuously irradiated from the side surface and the entire irradiation region is simultaneously observed by the two-dimensional detector, a large amount of light is received and high sensitivity is obtained.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION An example of an embodiment of the present invention will be described below with reference to FIG. FIG. 1 is a conceptual diagram of the device. The optical system is a sectional view. A 6% polyacrylamide gel plate 2 sandwiched between two 0.3 mm-spaced glass plates 1 (300 mm × 200 mm × 5 mm) is irradiated with an argon laser (400 nm 10 mw) from the side. The irradiation unit 3 is represented as a dot in the sectional view. The light emitted upward from the irradiation unit is refracted by the upper prism 4, and then the lens 6
Then, an image is formed on the lower side of the two-dimensional detector 9. On the other hand, the light emitted to the lower portion is refracted by the lower prism, and then also imaged on the upper side of the two-dimensional detector 9 by the lens 6.

In this embodiment, the DNA fragment as a sample for fluorescence detection is a fluorescent dye having four different emission wavelengths corresponding to four terminal base species, that is, FITC (fl
uorescein isothiocyanate; emission wavelength 515 nm), N
BD-F (4-fl-uoro-7 nitrobenzofurazan; emission wavelength 540 nm), TRITC (tetramethyl rhodamine is
othiocyanate; emission wavelength 573 nm) and Texas
Since it is fluorescently labeled with Red (emission wavelength 610 nm), the light of the above four wavelengths is emitted from the light emitting part, and these are dispersed by the prism, and as shown in FIG. Lines (4 points in cross section) are created. 15 and 15 ', 16 and 16', 17 and 17 ', 18
Reference numerals 18 'and 18' respectively represent images of light of the same wavelength, which correspond to light passing through the lower and upper prisms. In this case 15,
15 'is light from Texas Red, 16 and 16' are TRITC, 17 and 17 'are NBD-F, 18 and 18'.
Is an emission image from FITC.

In this example, the wavelength difference between 15 and 16, 17 and 18 is small, and it is difficult to sufficiently discriminate by wavelength dispersion by the prism, but 15 and 17 and 16 and 18 can be discriminated sufficiently. Therefore, by using a bandpass filter having a transmission band at two positions of 515 nm and 573 nm in the upper part 7 and a filter having a transmission band at two positions of 540 nm and 610 nm in the lower part, four colors can be distinguished and detected.

FIG. 2 shows this state. When there is no filter (excitation light removal filter is provided), eight lines are observed on the two-dimensional detector, and adjacent lines are not clearly separated. 4 with filters 7 and 8
The lines of the book can be detected separately (see the upper right part of FIG. 2 and the monitor screen of FIG. 1). The signals of the four fluorescent images can be read out independently by using one or two horizontal scanning lines of the two-dimensional detector. The obtained signal is converted into information on four types of terminal base species in the DNA fragment by using the data processor 11, and the base sequence is determined.

In this embodiment, the prisms 4 and 5 are integrated to have a narrow apex angle of 30 °. Ba for the prism material
F glass was used. The apex angle of the prism is determined so that light passing through the thick portion of the prism 4 passes below the lower end of the lens 6 by refraction, and light corresponding to a solid angle at which the lens sees an image can enter the lens.

FIG. 3 shows an example using a polyhedral prism.
The light emitted from the light emitting point 3 passes through the upper prism or the lower prism and is imaged at another position on the two-dimensional detector 9 by the lens 6. When the upper prism and the lower prism are composed of two surfaces with different angles as shown in the figure, the light passing through the upper and lower prisms is imaged on the detector 9 as two images, respectively, that is, four images in total. . Although the number of images increases, the light receiving solid angle seen at the prism also becomes large as a whole, so the amount of light received per detected fluorescent image hardly changes as compared with the case where no prism is used. Filters 20 having different transmission wavelengths are placed immediately after the image forming positions of the respective images or the prisms (4, 5), and the signals are detected by wavelength separation. Since the bandpass filter can sharply cut off the transmittance depending on the wavelength, it is possible to perform wavelength separation with higher accuracy than using wavelength dispersion by a prism. BK5 was used as the prism material in the above embodiment. The prism angles 21, 22 are 10 ° and 20 °, respectively.

[0024]

According to the present invention, a plurality of fluorescent images are first formed by using a prism, and then each image is formed on a light receiving device through a filter which distinguishes and transmits light having different wavelengths. Compared with 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 drawings]

FIG. 1 is a diagram illustrating a method of the present invention.

FIG. 2 is a schematic sectional view of the optical system in FIG.

FIG. 3 is a conceptual diagram of a measurement system using a polyhedral prism. [Brief Description of Drawings] 1 ... Glass plate, 2 ... Gel plate, 3 ... Irradiation part, 4 ... Upper prism, 5 ... Lower prism, 6 ... Lens, 7 ... Upper filter, 8 ... Lower filter, 9 ... Two-dimensional Detector, 10 ...
Control device, 11 ... Data processing device, 12 ... Display device, 1
3 ... Monitor, 14 ... Line image, 15, 15 'to 18, 1
8 '... fluorescence image, 19 ... excitation light cut filter, 20 ...
4-stage split bandpass filter 21,22 ... Prism angle

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Keiichi Nagai 1-280, Higashi Koigokubo, Kokubunji, Tokyo Inside the Central Research Laboratory, Hitachi, Ltd.

Claims (2)

[Claims] 1. A fluorescence detection method in a fluorescence detection type electrophoretic apparatus, wherein samples labeled with different fluorescent substances are electrophoretically separated in a plurality of migration paths, and then detected by irradiating laser light. From a predetermined position to irradiate the plurality of migration paths with laser light, excite the phosphor, and form a light emission line image along a path irradiated with the laser light; Dividing into a plurality of images, selecting a wavelength in each optical path of the light of the divided plurality of images, and forming a plurality of the divided images on a photodetector. A method for detecting fluorescence in a fluorescence detection type electrophoresis apparatus, which comprises detecting fluorescence from the phosphor that labels the sample separated by electrophoresis. 2. A fluorescence detection method in a fluorescence detection type electrophoretic device, wherein samples labeled with different fluorophores are electrophoretically separated in a plurality of migration paths and then detected by irradiating with laser light. From a predetermined position, irradiating a laser beam so as to penetrate the plurality of migration paths from a direction intersecting with the migration path, and forming a light emission line image along the path irradiated with the laser, Dividing the emission line image into a plurality of images; selecting a wavelength in each optical path of the light of the plurality of divided images; forming a plurality of the divided images on a photodetector; The method for detecting fluorescence in a fluorescence detection type electrophoretic device, comprising detecting fluorescence from the phosphor that labels the sample that has been electrophoretically separated.