JP2902408B2 - Fluorescence detection type electrophoresis device - Google Patents

Description

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

[Conventional technology]

DNA fragments with different terminal bases are labeled with fluorophores with different emission wavelengths, and DNA that passes through the irradiating part based on changes in fluorescence wavelength and intensity emitted by irradiating light at a certain position in the migration path while performing gel electrophoresis separation Techniques for determining the terminal base type of a fragment and determining the sequence have been developed. For the identification and detection of the fluorescence wavelength, a rotating filter with four kinds of bandpass filters is used in front of the photomultiplier tube, or two photomultiplier tubes with filters having different transmission wavelength bands are used. doing. In each case, the detector was swept across several migration paths to determine the base sequence of multiple sample DNA, while laser light was incident from the side of the gel plate, and each measurement point was continuously irradiated, A method has been proposed in which the obtained fluorescent image is spectrally separated by a prism and detected by a two-dimensional detector.

However, in the measurement system that sweeps the detector, the ratio α of the measurement time per one measurement point of the gel is as follows, where the length of the measurement area is 1 and the width of the irradiation laser beam is d. Becomes Usually d is 0.2-0.3mm, l100mm, so α10
^-3 , and only about 10 ^{-3 of the} amount of received fluorescence was obtained when continuous light irradiation and light reception were performed, and there was a problem that high sensitivity could not be obtained. On the other hand, in the system in which the light is separated by the prism and detected by the two-dimensional detector, the amount of received light is large, and this difficulty can be overcome. However, the spectroscopic accuracy of the prism is low, and there is a problem in highly accurate base identification.

[Problems to be solved by the invention]

As described above, the prior art does not consider the point of increasing the sensitivity, or does not consider the high-precision wavelength separation, and cannot determine the base sequence with high accuracy.

The object of the present invention is to solve the above-mentioned difficulties, and to achieve high precision and high sensitivity.
An object of the present invention is to provide a device capable of detecting and separating DNA.

[Means for solving the problem]

As a result of the study, the present inventors first divided a fluorescence image obtained by laser irradiation on the gel electrophoresis separation plate into a plurality of virtual images by a prism, and then band-divided the light of each of the image-divided images. Through a process such as wavelength dispersion by a pass filter, these images are formed on a detector to perform required separation and detection, and it has been found that the above-mentioned object is satisfactorily achieved. Further research based on the findings led to the completion of the present invention.

Therefore, the fluorescence detection type electrophoresis apparatus of the present invention is a fluorescence detection electrophoresis apparatus including at least a laser light source, a gel electrophoresis separation plate, and a fluorescence detector. A prism having a plurality of optical planes for dividing the obtained linear fluorescent 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. It is characterized by having.

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 optical system is provided with, in the light path of the divided image emerging from the plurality of optical planes of the prism, respectively different band-pass filters each having a correspondingly installed transmission wavelength band. .

As a specific shape of the prism, there is a polyhedron symmetrical in cross section including a ridge of a vertex at the center as shown in the drawings of the embodiment, or a polyhedron symmetrical in cross section at the center such as a trapezoid.

And, as the material of the prism, particularly when the device of the present invention is used as a multicolor fluorescence detection type electrophoresis device, it is necessary to use a material having a high refractive index, specifically, BaF01, Laf3, SF3, glass and the like can be mentioned.

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 this case includes FITC (fluorescein isothiocyanate; emission wavelength 515 n
m), NBD-F (4-fluoro-7nitrobenzofurazan; emission wavelength 540 nm), TRITC (tetramethyl rhodamine isothiocyan
ate; emission wavelength 573nm) and Texas Red (emission wavelength 610n)
m) or a phosphor containing a metal complex can be used.

As a bandpass filter corresponding to the multicolor mark, a combination of a dielectric vapor-deposited multilayer filter and a color glass filter is used.

In addition, as a desirable arrangement of the prism, at least one apex angle of the prism is such that the emission line image of the linear fluorescence image obtained by irradiating a laser beam to a required portion of the electrophoresis separation plate and the image formation site of the fluorescence detector. A line which is located in a plane including the center and is parallel to the emission line image of the fluorescence image, or the prism is a line obtained by irradiating a required portion of the gel electrophoresis separation plate with laser. The emission line image of the fluorescent image is vertically symmetrical with respect to a plane including the center of the imaging site of the fluorescence detector, and the apex angle of the center is parallel to the emission line image of the fluorescence image. Installed in

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 target samples.

The device of the present invention is most preferably used as a multicolor fluorescence detection-type electrophoresis device as described above, but can also be used for single-color fluorescence detection. Also in this case, using the apparatus of the present invention, the linear fluorescent image is divided into two images, for example, and the peak fluorescence light is measured by the former, and the light of a specific low wavelength is measured by the latter. By selecting a combination of band-pass filters, there is an advantage that it is possible to correct a measurement error due to a change in measurement conditions such as fluctuations of laser irradiation light of the apparatus and to perform accurate separation detection. .

[Action]

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

More specifically, in the fluorescence detection type electrophoresis apparatus of the present invention, when the irradiation part is viewed from the pupil position of the light receiving lens, the apex angle of the center of the prism preferably overlaps the linear light emission part. It has been placed. The light passing through the upper half and the lower half of the prism forms two images on the two-dimensional detector like light emitted from different points, and causes chromatic dispersion in the vertical direction. Light coming from each prism passes through a different filter and reaches the detection unit. Signals with similar wavelengths are separated and detected depending on whether they have passed through the upper half or lower half of the prism. Can be detected by chromatic dispersion. According to the present invention, fluorescence having different emission wavelengths can be separated and detected with high accuracy without time-divided detection, that is, without detecting time-dependently by combining a prism and a filter.

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. FIG. 1 is a conceptual diagram of the apparatus. The optical system is a sectional view. Two glass plates 1 with 0.3mm spacing (300mm × 200mm × 5m
m), the 6% polyacrylamide gel plate 2 is irradiated from the side with an argon laser (400 nm, 10 mw).
The irradiation unit 3 is represented as a point in the sectional view. The light emitted upward from the irradiating section is refracted by the upper prism 4 and forms an image on the lower side of the two-dimensional detector 9 by the lens 6. On the other hand, the light emitted to the lower part is refracted by the lower prism and then forms an image on the upper side of the two-dimensional detector 9 by the lens 6. In this embodiment,
The DNA fragment, which is a sample for fluorescence detection, has four types of fluorescent dyes having different emission wavelengths corresponding to four types of terminal bases, ie, FI
TC (fluorescein isothiocyanate; emission wavelength 515nm), NBD
-F (4-fluoro-7nitrobenzofurazan; emission wavelength 540n
m), since it is fluorescently labeled with TRITC (tetramethyl rhodamine isothiocyanate; emission wavelength 573 nm) and Texas Red (emission wavelength 610 nm), the light of the above four wavelengths is emitted from the emission part, and these are dispersed by the prism. As shown in FIG. 2, four lines (four points in a sectional view) are formed at the upper part and the lower part, respectively. 15 and 15'16 and 16 ', 17 and 17', 18
And 18 'are images of light of the same wavelength, respectively, corresponding to the light passing through the lower and upper prisms. In this case 15,15 '
Is light from Texas Red, 16, 16 'is TRITC, 17, 17' is NBD
-F, 18, 18 'are emission images from FITC. In this example, the wavelength difference between 15 and 16, and 17 and 18 is small, and it is difficult to identify sufficiently by the chromatic dispersion by the prism.
And 18 are sufficiently distinguishable. Therefore, by using a filter having transmission bands at two places of 515 nm and 573 nm in the upper part 7 and a filter having two transmission bands of 540 nm and 610 nm at the lower part, four colors can be distinguished and detected.
FIG. 2 shows this state. In the case where no filter is provided (the excitation light removing filter is provided), eight lines are observed on the two-dimensional detector, and adjacent lines are not clearly separated. When the filters 7 and 8 are mounted, the four lines can be detected separately. (Fig. 2 upper right and first
(Refer to the monitor screen in the figure.) The signals based on the four fluorescent images can be read out independently using one or two horizontal scanning lines of the two-dimensional detector. The resulting signal is processed by a data processor
The information is converted into information on the four types of terminal bases in the DNA fragment using 11 and the base sequence is determined.

In this embodiment, the prisms 4 and 5 are integrated to form a narrow vertical angle of 30 °.
And Baf glass was used for the prism material. The vertex 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 due to refraction, and light corresponding to a solid angle at which the lens views an image can enter the lens.

FIG. 3 shows an example using a polyhedral prism. Light emitting point 3
The light emitted from the lens 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 formed of two surfaces having different angles as shown in the figure, light passing through the upper and lower prisms forms two images on the detector 9 as a total of four images. Although the number of images increases, the light receiving solid angle seen at the prism also increases as a whole, so that the amount of received light per detected fluorescent image hardly changes as compared with the case where no prism is used. A filter 20 having a different transmission wavelength is placed immediately after the image formation position of each image or immediately after the prism (4, 5), and the signals are detected by wavelength separation.
The bandpass filter can sharply cut the transmittance due to the wavelength, so that the wavelength separation can be performed with higher accuracy than using the wavelength dispersion by the prism. Note that BK5 was used as the prism material in the above embodiment. The angles 21, 22 of the prism are 10 ° and 20 °, respectively.

〔The invention's effect〕

According to the present invention, a plurality of fluorescent images are first formed using a prism, and then each image is formed on a light receiver through a filter that identifies and transmits light having different wavelengths. As compared with the method of separation and detection by the method described above, signals from multicolor-labeled DNA fragments and the like can be distinguished with high accuracy and simultaneously measured, and separation and detection or base sequence determination with high accuracy can be performed.

[Brief description of the drawings]

FIG. 1 is a block diagram including a schematic sectional view of a measurement system using the fluorescence detection type electrophoresis apparatus of the present invention, FIG. 2 is a schematic sectional view of an optical system, and FIG. 3 is a schematic view of a measurement system using a polyhedral prism. It is a conceptual diagram. 1 ... glass plate, 2 ... gel plate, 3 ... irradiation unit, 4 ...
Upper prism, 5: Lower prism, 6: Lens, 7
... upper filter, 8 ... lower filter, 9 ... two-dimensional detector, 10 ... controller, 11 ... data processor,
12 ... display device, 13 ... monitor, 14 ... line image, 15,1
5 'to 18,18': Fluorescent image, 19: Excitation light cut filter, 20: 4-stage split bandpass filter, 21, 22 ...
The vertex angle of the prism.

 ────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Keiichi Nagai 1-280 Higashi Koigakubo, Kokubunji-shi, Tokyo Inside the Central Research Laboratory, Hitachi, Ltd. (56) References JP-A-209288 (JP, A) JP-A-52-37060 (JP, A) JP-A-63-19601 (JP, A) JP-A-59-187309 (JP, A) JP-A-63-231247 (JP, A) Japanese Patent Application Laid-Open No. Sho 63-36147 (JP, A) Japanese Patent Application No. Sho 62-93987 (Japanese Utility Model Application No. 64-3274), a microfilm (JP, U) photographing the contents of the specification and drawings attached to the application form

Claims (5)

(57) [Claims] 1. A plurality of migration paths each having a plurality of regions sandwiched between a first surface and a plurality of second surfaces having a different inclination from the first surface and arranged in one direction. A prism that divides a fluorescence image emitted from a phosphor that labels a sample that migrates a linear irradiation area irradiated with laser light almost simultaneously into a plurality of parallel line images, and the irradiation area and a photodetector. A plurality of bandpass filters having different transmission wavelength bands disposed therebetween, wherein the fluorescence image is detected at different positions of the photodetector as a plurality of parallel line images having different wavelength bands. Electrophoresis device. 2. A plurality of electrophoresis paths each having a plurality of regions sandwiched by a first surface and each of a plurality of second surfaces having a different inclination from the first surface and arranged in one direction. A prism that divides a fluorescence image emitted from a phosphor that labels a sample that migrates a linear irradiation area irradiated with laser light almost simultaneously into a plurality of parallel line images, and the irradiation area and a photodetector. A plurality of band-pass filters having different transmission wavelength bands disposed therebetween, wherein a ridge line of a vertex at a central portion of the prism includes the fluorescence image and the center of an image forming plane of the photodetector. Wherein the fluorescence image is detected as a plurality of parallel line images having different wavelength bands at different positions of the photodetector. 3. A plurality of electrophoresis paths on which a sample is electrophoresed, and each of a first surface and a plurality of second surfaces having a different inclination from the first surface are arranged in one direction. A plurality of regions, and a plurality of parallel lines are divided into a plurality of parallel line images by irradiating a linear irradiation region in which the plurality of migration paths are irradiated with laser light at the same time. And a plurality of bandpass filters having different transmission wavelength bands disposed between the irradiation area and the photodetector, wherein the first surface is substantially parallel to a surface formed by the plurality of migration paths. Wherein the fluorescence image is detected as a plurality of parallel line images having different wavelength bands at different positions of the photodetector. 4. A linear shape irradiated with a laser beam having four regions sandwiched by a first surface and four second surfaces having different inclinations from the first surface. A prism that divides a fluorescence image emitted from a phosphor that labels a sample migrating the irradiation region into four line images, and four bands having different transmission wavelength bands disposed between the irradiation region and the photodetector. A pass filter, and the ridgeline of the apex angle at the center of the prism is arranged in a plane including the fluorescence image and the center of the imaging plane of the photodetector, and the fluorescence image is in a wavelength band. An electrophoresis apparatus, wherein four different parallel line images are detected at different positions of the photodetector. 5. A plurality of migration paths on which a sample is migrated, and four regions sandwiched between a first surface and each of four second surfaces having an inclination different from that of the first surface. A prism that divides a fluorescence image emitted from a fluorescent substance that labels the sample, which migrates a linear irradiation area in which the plurality of migration paths are irradiated with laser light, into four line images, and the irradiation area And four bandpass filters having different transmission wavelength bands disposed between the photodetectors, wherein the first surface is disposed substantially parallel to a surface formed by the plurality of migration paths, and Is detected at different positions of the photodetector as four parallel line images having different wavelength bands.