JP2000338087A - Electrophoretic apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an electrophoretic apparatus capable of obtaining many information relative to each other in a short time by simplifying a migrating unit and obtaining an accurately migrating pattern. SOLUTION: The electrophoretic apparatus conducts an electrophoresis of a sample labeled by a fluorescent pigment in a migrating unit 201 and reads a luminescent fluorescent pattern. In this case, the apparatus comprises an electrophoretic device 200 for conducting an electrophoresis by feeding a plurality of samples for connecting different fluorescent pigments to a labeled substance such as many types of proteins or DNA or the like to the same lane of the unit 201, and a cofocal scanner or a fluorescent image measuring device for scanning the sample of the unit 201 by an exciting light and simultaneously detecting multicolor fluorescent pattern of the sample emitting by illuminating with an exciting light through a plurality of filters having different transmitting characteristics of multicolor fluorescent pattern of the sample emitted by irradiating with the light, thereby simultaneously detecting a plurality of types of migrating patterns.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoresis apparatus used in the field of biotechnology, and more particularly to an improvement for increasing the speed and resolution of electrophoresis.

[0002]

2. Description of the Related Art Hitherto, electrophoresis has been well known as a method capable of analyzing the structure of a gene or the structure of a protein such as an amino acid using an inexpensive and simple apparatus, and is widely used in the field of biotechnology. .

[0003] Electrophoresis includes disk electrophoresis using polyacrylamide, SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis, isoelectric focusing, nucleic acid gel electrophoresis, and others. There are electrophoresis, two-dimensional electrophoresis, and capillary electrophoresis utilizing the influence of interaction with molecules.

FIG. 9 is a block diagram showing an example of a conventional electrophoresis measuring device. This electrophoresis measuring device is roughly composed of an electrophoresis device section 10 and a signal processing device section 20. The electrophoresis device section 10 includes an electrophoresis section 11 that performs electrophoresis, a first electrode 12 and a second electrode 13 for applying a voltage to the electrophoresis section 11, and a support that holds the electrophoresis section 11 and the electrodes 12 and 13. A plate 14, an electrophoresis power supply 15 for applying a voltage to the electrodes, a light source 16 for generating light for exciting a fluorescent substance, and an optical fiber 17 for guiding light from the light source 16.
And a photodetector 18 for condensing fluorescence generated from a fluorescent substance, selectively passing light of a specific wavelength through an optical filter, and converting the light into an electric signal.

The signal processing unit 20 receives the electric signal from the photodetector 18 and performs appropriate processing such as pre-processing such as data conversion into digital data and averaging processing. Has become. The output of the signal processing unit 20 is sent to a data processing device (not shown), and the sample is analyzed by an analysis process.

In such an apparatus, a gel is injected into the electrophoresis section 11 and D is labeled with a fluorescent substance from above the gel.
When a sample of an NA fragment is injected, and subsequently a voltage is applied to the first electrode 12 and the second electrode 13 by the power supply device 15, electrophoresis starts. In each lane of the sample, the molecules contained in the sample are collected for each molecular weight, and a band is formed. The lighter the molecular weight, the faster the migration speed, and thus the longer the migration distance in the same time.

The detection of these bands is performed by irradiating the gel with light (eg, laser light) from the light source 16 to generate fluorescence in the labeled fluorescent substance collected in the band in the gel. Detected by the detector 18.

That is, when the laser beam is irradiated,
As shown in FIG. 0, the fluorescent substance of the gel existing on the optical path 31 is excited to emit fluorescence. This fluorescence is detected at a predetermined detection position for each lane in the electrophoresis direction over time. As a result, fluorescence is detected when the band 32 of each lane passes through a position on the optical path 31.
A pattern signal of the fluorescence intensity in one lane is obtained. A data processing device (not shown) can analyze, for example, each base sequence of DNA from the pattern signal.

[0009]

However, such a conventional electrophoresis apparatus has the following problems. Measurement takes time. Insufficient resolution. Many lanes are required for many types of separations. Since two dimensions are the limit, three or more dimensions of mutual relation information cannot be obtained. Large installation area. The area of the electrophoresis section is, for example, 50 c
Each of them is as large as mx 50 cm or 5 cm x 5 cm. Particularly in two dimensions, the reproducibility of the position is poor. It is sufficient to insert a marker in another lane and refer to it, but if the marker is inserted, the area increases.

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems. The electrophoresis section is compact, a high-precision electrophoresis pattern can be obtained, and a lot of related information can be obtained in a short time. An electrophoresis apparatus is provided.

[0011]

In order to achieve the above object, according to the first aspect of the present invention, a sample labeled with a fluorescent dye is electrophoresed in an electrophoresis section to read a fluorescent pattern to emit light. An electrophoresis apparatus unit for performing electrophoresis by flowing a plurality of samples each having a different fluorescent dye bound to a target substance such as various proteins or DNAs in the same lane of the electrophoresis unit. A confocal structure configured to scan the sample of the electrophoresis section with excitation light and simultaneously detect a multicolor fluorescent pattern of the sample emitted by irradiation of the excitation light through a plurality of filters having different transmission characteristics. A scanner or a fluorescence image measuring device is provided so that a plurality of types of migration patterns can be detected simultaneously.

According to such a configuration, the number of lanes is reduced, the electrophoresis section is made compact, and non-uniformity of the voltage gradient and non-uniformity of the gel can be prevented, so that highly accurate measurement can be performed. Further, a multicolor fluorescent pattern can be simultaneously detected by a confocal scanner or a fluorescent image measuring device, and the detection time can be reduced.

According to a second aspect of the present invention, there is provided an electrophoresis apparatus configured to electrophorese a sample labeled with a fluorescent dye in an electrophoresis section and read a fluorescent pattern that emits light. Flowing the target substance to the electrophoresis section,
An electrophoresis device unit for performing electrophoresis by providing a physical gradient such as voltage, pH, and density in the thickness direction of the sample, and the sample that scans the sample of the electrophoresis unit with excitation light and emits light by irradiation with excitation light A scanning or non-scanning confocal microscope or a two-photon excitation microscope configured to simultaneously detect the multicolor fluorescent pattern through a plurality of filters having different transmission characteristics. It is characterized in that a dimensional position and a density can be detected.

In such a configuration, even if electrophoresis is performed by providing a physical gradient such as voltage, pH, and density in the thickness direction of the sample, the electrophoresis can be performed simultaneously by using a confocal microscope or a two-photon excitation microscope. A three-dimensional migration pattern can be detected.

In the invention of claim 2, in this case, by providing different physical gradients in the plane direction and the thickness direction and performing electrophoresis simultaneously in the three axis directions, high speed and three axis Can measure the interrelationship of effects.

Further, when the sample and the marker are arranged side by side in the thickness direction of the sample as in claim 4, other conditions such as gel concentration, temperature and voltage distribution become the same, so that high accuracy is achieved. Compact measurement becomes possible.

Further, when the aperture of the confocal microscope is arranged so as to coincide with the position of each sample or to be located inside the position of the sample, a high S / S ratio free from the edge of the sample is provided. N can be measured.

If the light condensing means is provided on the light source side of the opening as in claim 6, the light source can be effectively used.

Further, the concentration distribution in the thickness direction can be realized by bringing a high-concentration solution into contact with only one side of the gel, or by providing a density difference in the thickness direction by centrifugation or lamination of multiple layers.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. FIG. 1 is a main part configuration diagram showing an embodiment of the electrophoresis apparatus according to the present invention. In the figure, 100 is a confocal microscope section, 2
Reference numeral 00 denotes an electrophoresis device.

The confocal microscope unit (also referred to as a confocal optical scanner) 100 is capable of optically scanning the carrier of the electrophoretic unit 201 and reading the electrophoretic pattern of the fluorescence emitted from the carrier. It has become.

The excitation light (for example, wavelength λ)
1 is converted into parallel light by a lens 102, reflected by a dichroic mirror 103, condensed on a slit of a slit array 105 via a lens 104, and subsequently passed through the slit by an objective lens 10.
6 and is incident on the carrier of the electrophoresis unit 201. The fluorescent substance in the migration section is excited by this light and emits fluorescence.

This fluorescence returns to the objective lens 106, the slit array 105, and the lens 104 again, passes through the dichroic mirror 103, and enters the next dichroic mirror 107. The dichroic mirror 10
3 reflects light having a wavelength of λ1 (for example, blue);
Transmits longer wavelength light. The dichroic mirror 107 reflects light of wavelength λ2 (for example, green),
Light of wavelength λ3 (for example, red) is transmitted. Wavelength λ1,
λ2 and λ3 have a relationship as shown in FIG.

The light of wavelength λ2 reflected by the dichroic mirror 107 is condensed on a light receiver 109 via a lens 108, and the light of wavelength λ3 transmitted on the dichroic mirror 107 is condensed on a light receiver 111 via a lens 110. . When the slit array 105 is moved to control the light from the light source to optically scan the surface of the migration unit 201, a fluorescent electrophoresis pattern generated by the migration unit 201 forms an image on each of the light receivers 109 and 111.

In this case, only the pattern which emits green light in the electrophoresis pattern is imaged on the light receiving
Only the pattern that emits red light in the migration pattern forms an image. The light receivers 109 and 111 convert the formed image into an electric signal and output it.

The electrophoresis device section 200 includes an electrophoresis section 201.
And a power supply 202 for supplying a voltage for performing electrophoresis in the migration section 201.

As described above, the multicolor fluorescent electrophoresis pattern generated in the electrophoresis section 201 can be easily measured with high precision using the confocal scanner.

Incidentally, the absolute value of the molecular weight cannot be obtained by electrophoresis. For this reason, a marker molecule for reference is usually supplied to an adjacent lane as shown in FIG. 3, but in this method,
There is a problem that a measurement error occurs due to a space requirement and difficulty in applying a voltage uniformly over all lanes.

In the present invention, as shown in FIG. 4, a marker molecule for reference (hereinafter simply referred to as a marker) is mixed with the sample and flows into the same lane. However, a dye having a different fluorescence wavelength is bound to each marker and sample. By electrophoresing such a substance and optically scanning with a confocal scanner, a plurality of types of fluorophore patterns can be detected simultaneously.

FIG. 5 shows another embodiment of the present invention. Although two-dimensional electrophoresis is generally well known, FIG. 5 shows an example of three-dimensional electrophoresis in which one dimension is added in the thickness direction (Z-axis direction).

In this case, for example, in the X-axis direction (vertical direction),
The following voltage gradient and pH gradient are applied to the Y-axis direction (lateral direction) and the Z-axis direction (thickness direction). 1) A high voltage is applied in the X-axis direction, a pH gradient is applied in the Y-axis direction, and a low voltage is applied in the Z-axis direction. 2) A multilayer gel having a voltage applied in the X-axis direction, a pH gradient in the Y-axis direction, and a gel concentration varied in the Z-axis direction. 3) A voltage is applied in the X-axis direction, a pH gradient is applied in the Y-axis direction, and a voltage gradient is applied in the Z-axis direction to perform affinity electrophoresis.

In this case, for example, the objective lens 106 of the confocal scanner 100 is configured to be able to move up and down so that the light scanning surface of the electrophoresis unit 201 can move up and down in the optical axis direction (Z axis direction). And while controlling the optical scanning surface in the Z-axis direction,
By performing multicolor detection of the fluorescent electrophoresis pattern in the XY axis directions, three-dimensional information can be easily obtained.

The foregoing description has been directed to specific preferred embodiments for the purpose of illustration and illustration of the invention. Therefore, the present invention is not limited to the above-described embodiments, and includes many more modifications without departing from the spirit thereof.
This includes deformation.

For example, in the embodiment of FIG. 5, if only XZ is used as a lane as shown in FIG. 6, for example, it becomes more compact than ordinary two-dimensional electrophoresis. The concentration distribution in the thickness direction (Z-axis direction) can be realized by contacting a high-concentration solution on one side only, or by providing a density difference in the thickness direction by centrifugation. Alternatively, it can be realized by a method in which gels having different concentrations are laminated in multiple layers.

When the sample and the marker are separately inserted in the thickness direction as shown in FIG. 7, other conditions are the same as in FIG. 6, and compact measurement can be performed. In this case, since the thickness direction can be measured separately by the confocal method, the fluorescent colors may be the same. Furthermore, as shown in FIG. 8, when electrophoresis is measured with a non-scanning confocal microscope, it is necessary to set the confocal opening 61 so as to coincide with the sample position 62 or to measure a part thereof. In addition, high S / N measurement without the influence of the edge of the sample can be realized.

It should be noted that either a single-photon or two-photon light source can be used as the light source, and the same effect can be obtained.

[0037]

As described above, according to the present invention, the following effects can be obtained. 1) Compact and highly accurate multicolor electrophoresis can be easily realized. 2) Three-dimensional electrophoresis can be realized, the apparatus is compact, and a large amount of correlated information can be obtained in a short time.

[Brief description of the drawings]

FIG. 1 is a configuration diagram showing one embodiment of a multicolor electrophoresis apparatus according to the present invention.

FIG. 2 is an explanatory diagram showing wavelength distributions of excitation light and fluorescence.

FIG. 3 is an explanatory diagram of an arrangement of a sample and a marker.

FIG. 4 is an explanatory diagram when a sample and a marker are injected into the same lane.

FIG. 5 is an explanatory diagram of a migration unit when performing three-dimensional migration.

FIG. 6 is an explanatory diagram in a case where one axial direction is separated.

FIG. 7 is an explanatory diagram when markers are arranged in the thickness direction of a sample.

FIG. 8 is an explanatory diagram showing a relationship between a position of a sample and an opening.

FIG. 9 is a configuration diagram illustrating an example of a conventional electrophoresis apparatus.

FIG. 10 is an explanatory diagram of an electrophoresis pattern.

[Explanation of symbols]

 REFERENCE SIGNS LIST 100 confocal microscope section 101 light source 102, 104, 108, 110 lens 103, 107 dichroic mirror 105 slit array 106 objective lens 109, 111 light receiver 200 electrophoresis apparatus section 201 migration section 202 power supply

Claims (7)

[Claims] An electrophoresis apparatus configured to electrophorese a sample labeled with a fluorescent dye in an electrophoresis section and read a fluorescent pattern that emits light, wherein the electrophoresis apparatus is different for various kinds of target substances such as proteins or DNA. An electrophoresis apparatus for performing electrophoresis by flowing a plurality of samples to which fluorescent dyes are bound in the same lane of the electrophoresis section; and the sample that scans a sample of the electrophoresis section with excitation light and emits light by irradiation with excitation light. Equipped with a confocal scanner or a fluorescence image measuring device configured to simultaneously detect the multicolor fluorescent pattern through a plurality of filters having different transmission characteristics, so that a plurality of types of migration patterns can be detected simultaneously. An electrophoresis apparatus, comprising: 2. An electrophoresis apparatus configured to electrophorese a sample labeled with a fluorescent dye in an electrophoresis section and read a fluorescent pattern to emit light, wherein a target substance such as various proteins or DNAs is applied to the electrophoresis section. An electrophoresis device section for performing electrophoresis by providing a physical gradient such as voltage, pH, density, concentration, etc. in the thickness direction of the sample, and scanning the sample of the electrophoresis section with excitation light, and irradiating with excitation light A scanning or non-scanning confocal microscope or a two-photon excitation microscope configured to detect the fluorescent pattern of the sample that emits light is provided so that the three-dimensional position and concentration of the sample can be detected. A three-dimensional electrophoresis apparatus, characterized in that: 3. The electrophoretic device according to claim 2, wherein
A three-dimensional multicolor electrophoresis apparatus, wherein the electrophoresis apparatus has different physical gradients in two plane directions and different thickness directions, and is configured to simultaneously separate samples in three axes. 4. The electrophoretic device according to claim 2, wherein
3. The three-dimensional electrophoresis apparatus according to claim 1, wherein the electrophoresis apparatus includes a sample and a marker provided in a thickness direction of the sample. 5. The electrophoretic device according to claim 2, wherein
The non-scanning confocal microscope is arranged so that an opening coincides with the position of each sample or is located inside the position of each sample. 6. An electrophoretic apparatus according to claim 1, wherein said confocal microscope has a condensing means on a light source side of said confocal opening. 7. The concentration distribution in the thickness direction according to claim 2,
An electrophoresis apparatus, which is realized by bringing a high-concentration solution into contact with only one side of a gel or providing a density difference in a thickness direction by centrifugation or lamination of multiple layers.