JP2982906B2 - Electrophoresis device - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an apparatus for measuring genes and nucleic acids, and more particularly to an electrophoresis apparatus capable of simultaneously measuring a plurality of pathogenic genes and a plurality of nucleic acid samples.

[Conventional technology]

Conventionally, the detection of a fluorescently labeled nucleic acid fragment has been performed by an electrophoresis apparatus shown in FIG. In this electrophoresis apparatus, a migration path 21 is provided in a migration gel 1 sandwiched between glass plates 20, and nucleic acid fragments are separated by migration. A laser 2 is applied from the side of the gel to irradiate all the electrophoresis paths at the same time, and the fluorescence from the nucleic acid labeled with the fluorescent substance is detected by the image amplification tube 3 and the one-dimensional array sensor 4, and data is processed by the computer 22.

The signal obtained in this manner is obtained as a waveform as shown in FIG. 11, and the base sequence of the nucleic acid can be determined by reading these peaks in time series.

According to this method, only one kind of nucleic acid sample can be analyzed in four migration paths.

Also, Science, 238 , pp. 336-341, '87
FIG. 12 shows an example of conventional four-color detection described in -10. In this example, four types of phosphors G-50 are used by using filters A and B of two colors.
5, A-512, C-519 and T-526 were detected. This method is 2
It is necessary that the wavelength range of the color filter includes the wave component of the emission spectrum of the phosphor. For this reason, when the types of phosphors are increased, there is a disadvantage that it becomes difficult to separate colors by two-color filters.

In the field of genetic engineering, the types and numbers of nucleic acids or genes to be analyzed tend to increase more and more in the future. Therefore, it has become necessary to perform multi-color detection in order to enable multiple types of analysis at the same time. Japanese Patent Application Laid-Open No. 63-231247 describes an electrophoresis apparatus in which four sample groups are labeled with different dyes, and these are put together and run on one migration path to separate them. In this apparatus, a sample to be electrophoresed is irradiated with a laser beam, and the fluorescence is condensed and measured by spectroscopy using a prism or a diffraction grating. For example, a luminescent image is collected by an imaging lens and split by a prism or diffraction grating to form an image on an image amplifier. Since the lights having different wavelengths are dispersed in the vertical direction on the image amplifier, their positions are shifted corresponding to the lights of the four colors, and appear on the two-dimensional detector via the coupling lens. That is, the apparatus determines the arrangement by detecting the time change of the signals corresponding to the four dyes, assuming that the position and intensity of the sample change with time. Therefore,
In this device, even if a plurality of samples are present at the same position, even if they are labeled with different phosphors, a plurality of types of fluorescence cannot be distinguished from each other. For this reason, in this apparatus, the emission wavelength of fluorescence is wide, and the emission of the dyes used, FITC and NBD-F, and the emission from TRITC and Texas Red cannot be completely separated. The data must be corrected to remove the contribution from the phosphor.

[Problems to be solved by the invention]

The above conventional method does not take into account the simultaneous analysis of various types of nucleic acids and genes, and has a problem in the configuration of the detector.

SUMMARY OF THE INVENTION It is an object of the present invention to improve the analytical processing performance of an electrophoresis apparatus by detecting multiple colors.

[Means for solving the problem]

In order to achieve the above object, the present invention uses the following two technical means. The first is that a nucleic acid sample is labeled with two or more kinds of fluorophores having different fluorescence wavelengths. Second, a detector that color-separates two or more types of fluorescence is used.

It is preferable to use the following detector as the detection means for color separation.

(1) A detector composed of a beam splitter, a color separation filter, and a photoelectric conversion element (2) A detector composed of a line-shaped filter and a photoelectric conversion element (3) A detector composed of a dichroic mirror and a photoelectric conversion element (4) Detector consisting of a color separation filter and a secondary electron multiplier (5) Detector consisting of a matrix filter and a two-dimensional photoelectric conversion element [Function] A nucleic acid sample labeled with a plurality of fluorescent substances is excited by a laser. Since the fluorescence spectra to be obtained are different, they can be analyzed simultaneously by color separation. Further, since the beam splitter and the separation filter color-separate a plurality of different fluorescence spectra, a plurality of nucleic acid samples can be simultaneously analyzed. Further, even if a matrix filter or a dichroic mirror is used, color separation of a plurality of different fluorescence spectra is possible.

By using a two-dimensional television camera for the matrix filter and the image intensifier, a plurality of fluorescence spectra can be separated as an image.

EXAMPLES Hereinafter, one example of the present invention will be described.

FIG. 1 shows a cross section of the electrophoresis gel 1, in which a nucleic acid sample 5 is fragmented by electrophoresis and passes through a laser irradiation area. Since the nucleic acid sample 5 is prepared by labeling four kinds of fluorescent substances for each sample, the nucleic acid fragment emits fluorescence when passing through the laser irradiation region. This fluorescence is applied to prism 6
And split the fluorescence into filters F1, F2, and F3, respectively.
Through. Since these fluorescences are extremely weak, they are amplified by the image intensifier tube 3 and are
It was converted into an electric signal by the dimensional array sensor 4. This output was subjected to data processing by a computer 11 via an interface 10.

Here, assuming that the outputs of the three array sensors are Ex, Ey, and Ez, the colors can be quantified by the following equations.

Here, x, y: chromaticity values expressing colors in two dimensions. Fluorescence spectra A1 to A4 of the phosphors (four types) used in this embodiment and spectral transmittances F11, F12, and F4 of color separation filters F1 to F3.
F13 is shown in FIG. From the fluorescence spectrum Aλ (λ: wavelength) and the spectral transmittance Fλ, the output E of the detector can be calculated by the following equation.

E = ∫λAλFλdλ (2) From this equation (2) and the above-mentioned equation (1), each phosphor A1
The chromaticity values x and y of .about.A4 are as shown in FIG.

As a result, four types of fluorescence spectra could be separated by the color separation filter. Further, the shape of the prism 6 is changed to the fourth
Shown in the figure. The fluorescent light incident on the prism 6 is refracted in three directions and outputted from the surfaces 6 ', 6 ", 6. These form three images when viewed from the image intensifier tube 3.
The above-mentioned color separation filters F1, F2, and F3 were attached to the emission side to enable color separation of incident fluorescence.

Here, since the fluorescence to be detected is weak, the image intensifier tube 3
In amplified, but the amplification degree is required for those of ¹⁰⁶ or more according to experiments. Then, the amplified photocurrents are arrayed in a CCD (Charge Coupled Device: Char) in every three filters.
ge Couple Device). This signal is amplified by the interface 10, quantized to a digital signal, and output to the computer 11. The computer 11 uses the above-described equation (1)
The color of fluorescence from the input data Ex, Ey, Ez was calculated.

An embodiment according to the present invention will be described with reference to FIG. In this example, a line-shaped filter 7 was used for color separation, and an image intensifier tube 3 and a two-dimensional television camera 8 were used. An example of a line-shaped filter is shown in FIG.
This is to separate incident fluorescent light by color filters F1, F2, F3. This output was amplified with an image by the amplifier tube 3 and further converted into a video signal by a two-dimensional television camera 8. Thereafter, the fluorescence color of this video signal could be detected by using the equation (1) in the same manner as in the embodiment of FIG.

Further, an embodiment according to the present invention will be described with reference to FIG.
This color used dichroic mirrors M1 and M2 for color separation. First, the mirror M1 was a dichroic mirror whose reflected light provided the spectral characteristics F13 shown in FIG. The mirror M2 is a dichroic mirror whose reflected light has a spectral characteristic of F11 in FIG. 2 and transmitted light has a characteristic of F12. The mirrors M3 and M4 were constituted by total reflection mirrors. Here, the length of the optical path is equal to F21 and F23, and F22 is shorter than the others. However, there is no problem if the depth of focus of the lens 1 is increased. This allows the fluorescence F0 from the sample to
The light was color-separated into components F21 to F23, and was incident on the image intensifier tube 3.

In the above description (FIGS. 1, 4 and 7), an image intensifier was used to amplify weak fluorescence.
An embodiment using a secondary electron multiplier in place of this is described in the eighth embodiment.
Shown in the figure. This is the color separation output F21, F22,
F23 is configured to receive light by each of the secondary electron multipliers P1, P2, and P3. According to this embodiment, the configuration of the apparatus can be simplified as compared with the embodiment in which the image amplification tube and the array sensor are combined.

The above embodiments have described a method for one-dimensionally (waveform) color-separating a plurality of nucleic acids. FIG. 9 shows an embodiment in which the present invention is applied to two dimensions (images). The three color filters F1, F2, and F3 according to the present invention were arranged in a matrix, and were closely attached to the image intensifier tube 3. Further, a two-dimensional television camera (for example, a CCD) was arranged on the output side of the image intensifier tube. As a result, it has become possible to detect patterns of nucleic acids and genes labeled with multicolor fluorescent substances.

In the above description, the case where the sample to be analyzed is a nucleic acid has been described. In addition, the sample can be applied to a gene or a protein.
Further, although the embodiment of the color separation has been described for four types of phosphors, it is apparent that the present invention can be applied to the detection of various types of phosphors within the range of the chromaticity values shown in FIG.

As described above, according to the present embodiment, it is possible to detect a fluorescent substance labeled on a plurality of nucleic acid or gene samples.

〔The invention's effect〕

As described above, according to the electrophoresis apparatus of the present invention, it is possible to simultaneously analyze a plurality of nucleic acid or gene samples, and there is an effect that the processing capacity is improved.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a configuration diagram of a color separation detection system using a beam splitter according to the present invention, FIG. 2 is a spectral transmittance spectrum of three kinds of color separation filters of the present invention, and FIG. FIG. 4 is a diagram showing the shape of the beam splitter according to the present invention, FIG. 5 is a block diagram of the matrix filter and the television camera according to the present invention, and FIG. 6 is a diagram showing the matrix filter according to the present invention. FIG. 7, FIG. 7 is a diagram showing a configuration of a dichroic mirror according to the present invention, FIG. 8 is a diagram showing a configuration in which a secondary electron multiplier is applied to the present invention, and FIG. 9 is a matrix filter and a two-dimensional filter according to the present invention. FIG. 10 is an explanatory diagram of a conventional electrophoretic device, FIG. 11 is an explanatory diagram of data obtained by a conventional electrophoretic device, and FIG. 12 is an explanatory diagram of a conventional color separation method. . 5 electrophoretic fragments of nucleic acids or genes, 6 prisms, F1, F2, F3 separation filters of three colors, 4 one-dimensional array sensors, 7 color filters of line shapes, M1, M2
... dichroic mirrors, P1, P2, P3 ... secondary electron multipliers, 8 ... television cameras, 11 ... computers.

──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Keiichi Nagai 1-280 Higashi Koikebo, Kokubunji-shi, Tokyo Inside the Central Research Laboratory, Hitachi, Ltd. (72) Inventor Seiko Hayasaka 2-6-1 Otemachi, Chiyoda-ku, Tokyo Hitachi (56) References JP-A-63-231247 (JP, A) JP-A-61-292027 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) G01N 27 / 447

Claims (9)

(57) [Claims] A plurality of electrophoresis paths for migrating a sample labeled with four kinds of fluorescent substances; and a laser beam simultaneously irradiating the plurality of electrophoresis paths, wherein the plurality of electrophoresis paths pass through a portion irradiated with the laser light. In an electrophoresis apparatus including light detection means for detecting fluorescence from the phosphor labeled on a sample, the light detection means comprises a wavelength separation means for separating and detecting the fluorescence into light in three wavelength regions. A photoelectric conversion unit that detects the fluorescence separated by the wavelength separation unit; and a unit that obtains a chromaticity value of each fluorescence from the four types of phosphors from the fluorescence detected by the photoelectric conversion unit. An electrophoresis apparatus, comprising: identifying each fluorescence from the four types of the phosphors. 2. The electrophoretic device according to claim 1, wherein
The wavelength separation means includes a prism that splits the fluorescence into three, and a line filter in which three filters that split the split fluorescence into light in the wavelength region are arranged one-dimensionally. Electrophoresis device. 3. The electrophoretic device according to claim 2, wherein
The electrophoresis apparatus according to claim 1, wherein the line filter includes a plurality of sets each including the three filters. 4. The electrophoretic device according to claim 1, wherein
The wavelength separating means includes a prism that divides the fluorescence into three, and a plurality of sets in which a set of three filters is two-dimensionally arranged, and separates the divided fluorescence into light in the wavelength region. An electrophoresis apparatus, comprising: 5. A plurality of migration paths for migrating a sample labeled with a first plurality of types of fluorescent substances, and simultaneously irradiating the plurality of migration paths with laser light, and irradiating the laser light. An electrophoresis apparatus including light detection means for detecting fluorescence from the fluorescent substance labeled on the sample passing through the site, wherein the light detection means reduces the fluorescence to less than the first plurality. Wavelength separating means for separating and detecting light in a second plurality of wavelength regions, photoelectric converting means for detecting the fluorescent light separated by the wavelength separating means, and fluorescent light detecting by the photoelectric converting means A means for determining a chromaticity value of each fluorescent light from the first plurality of types of fluorescent materials, and identifying each fluorescent light from the first plurality of types of fluorescent materials. Electrophoresis device. 6. The electrophoretic device according to claim 5, wherein
The wavelength separating means includes a prism that divides the fluorescent light into the second plurality, and a second plurality of filters that divide the split fluorescent light into light in the wavelength region, are arranged one-dimensionally. An electrophoresis apparatus, comprising: 7. The electrophoretic device according to claim 6, wherein
The electrophoresis apparatus according to claim 1, wherein the line filter includes a plurality of sets each including the second plurality of filters. 8. The electrophoresis apparatus according to claim 5, wherein
The wavelength separation unit has a prism that divides the fluorescence into the second plurality, and a plurality of sets of the second plurality of filters that are two-dimensionally arranged. An electrophoresis apparatus, which separates fluorescence into light in the wavelength region. 9. The electrophoretic device according to claim 5, wherein the first plurality is four and the second plurality is three. Electrophoresis device.