JPH07134101A - Dna base sequence determining apparatus - Google Patents

Abstract

PURPOSE:To transmit only fluorescent light effectively and surely to a fluorescent light detecting means by putting a P light deflecting plate between a fluorescent light detecting means and an electrophoresis plate and cutting excited light. CONSTITUTION:A fluorescent light detecting means is composed of a converging lens 10 and a light receiving element 12 and a P light deflecting plate 14 is put between the converging lens and an electrophoresis plate 74. The position of the light deflecting plate 14 may be between the lens 10 and the light receiving element 12. The excited laser to be used is linearly deflected laser light with 488nm wavelength and the scattered light at the time the laser light passes a gel electrolytic layer 86 has characteristics as excited light and contains a large quantity of P deflected components. Consequently, only fluorescent light with 520nm wavelength close to that of the laser light can be supplied to the light receiving element 12 by putting the P light deflecting plate 14, which transmits only P deflected light components and cut S deflected light components, and cutting the S deflected light components.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a DNA base sequencer. More specifically, the present invention uses D
The present invention relates to an apparatus capable of efficiently and quickly determining the base sequence of NA.

[0002]

2. Description of the Related Art Gel electrophoresis is widely used as a method for determining the base sequence of DNA or the like.

Conventionally, during electrophoresis, a sample was labeled with a radioisotope and analyzed, but this method has a drawback that it takes time and labor. Furthermore, from the viewpoint of radioactivity control, maximum safety and control are always required, and analysis can be performed only in a special facility. For this reason,
Recently, a method of labeling a sample with a phosphor has been studied.

In the method using light, fluorescently labeled DN is used.
The A fragment is electrophoresed in the gel.
A photoexcitation unit and a photodetector are provided for each migration path below -20 cm, and the DNA fragments passing therethrough are sequentially measured. For example, when a DNA chain whose sequence is to be determined is used as a template and an enzymatic reaction (dideoxy method) is performed, the number of terminal base species is reduced to 0.
Different lengths of DNA of different lengths are replicated and these are labeled with a fluorophore. That is, adenine (A) labeled with a fluorescent substance
A fragment group, a cytosine (C) fragment group, a guanine (G) fragment group, and a thymine (T) fragment group are obtained. These fragments are mixed and injected into separate migration lane grooves of the electrophoresis gel,
Apply voltage. Since DNA is a chain polymer having a negative charge, it moves in the gel at a speed inversely proportional to the molecular weight. Since shorter (smaller molecular weight) DNA chains move faster and longer (larger molecular weight) DNA chains move slowly, DNA can be fractionated by molecular weight.

In Japanese Patent Laid-Open No. 63-21556, a line on the gel of the electrophoretic device irradiated with a laser and the arrangement direction of the photodiode array are perpendicular to the electrophoretic direction of the DNA fragments in the electrophoretic device. The DNA nucleotide sequencer having the above-mentioned structure is disclosed.

FIG. 4 is a schematic diagram for explaining the structure of the apparatus. The electrophoresis plate 74 has an electrophoresis gel (for example, polyacrylamide) sandwiched between two glass plates. The overall thickness of the electrophoretic plate is about 10 mm, but the thickness of the gel electrolyte layer itself is less than about 1 mm. An upper end of the comb-shaped gel electrolyte layer exists at a position slightly lower than the upper end of the migration plate 74. DN labeled with a fluorescent substance in the comb-shaped groove 75
Inject the A fragment.

In the apparatus shown in FIG. 4, the laser light emitted from the light source 70 is reflected by the mirror 72, and a certain point in the gel of the electrophoretic plate 74 is horizontally irradiated with the laser light. When the fluorescently labeled DNA fragment 76 that has migrated in the gel passes through this irradiation region, fluorescence is sequentially emitted from this DNA fragment. At this time, the type of terminal base can be identified from the horizontal position of fluorescence emission, the fragment length can be identified from the difference in migration time from the migration start, and the analyte can be identified by the emission wavelength.
The fluorescence from each migration path is imaged by the light receiving portion 82 of the image intensifier 80 by the lens 78. This signal is amplified, converted into an electric signal by the photodiode array 84, and measured. The sequence of each DNA fragment is calculated by computer processing the measurement results, and the target DN is obtained.
The base sequence of A is determined.

Although the apparatus shown in FIG. 4 uses an image intensifier camera as a light receiving system, the image intensifier camera is not only an extremely expensive optical apparatus but also a relatively large apparatus. . Therefore, as shown in FIG. 5, a device has been developed in which the fluorescence detection means is individually arranged at the detection position of each migration path 88. Each fluorescence detecting means is composed of a filter 100, a condenser lens 110, and a light receiving element 120. The filter 100 is for removing excitation light and background light and selectively passing only fluorescence, and the condenser lens 110 is for focusing the fluorescence filtered by the filter on the light receiving surface of the light receiving element 120. Is used for. The light receiving element 120 is, for example, a photodiode made of silicon having a pn junction. Reference numeral 86 indicates a gel electrolyte layer.

For example, in such a DNA base sequencer, when an argon ion laser is used as a laser light source, the wavelength of laser light is about 488 nm.
Further, as a fluorophore for labeling a DNA fragment, FI
When TC is used, when the phosphor is irradiated with the laser light, fluorescence having a wavelength of about 520 nm is generated from the phosphor. Therefore, the filter 100 has the wavelength of 520n.
It is preferable that only the fluorescence of m can be transmitted and stray light of other wavelengths or background light can be removed.
Such a filter is, for example, a dielectric multilayer coating filter.

However, as shown in FIG. 6, since the wavelength of the excitation light and the wavelength of the phosphor are close to each other, it is actually necessary to manufacture a filter that can reliably and effectively cut only the wavelength of the excitation light. Very difficult. In the case of a dielectric multilayer coating filter, when a thin film having a low refractive index and a high refractive index is formed into a layer and is regarded as an equivalent film, a so-called Fabry-Perot Similar to the interferometer, the desired transmission is set by setting the film thickness and refractive index so that the optical path length is m · λ / 2 (m is an integer) with respect to the storage.
Only the light of the desired wavelength can be transmitted. But,
In manufacturing this multilayer film, it is necessary to increase the number of layers in order to improve the cut performance in a narrow band with respect to a desired transmission wavelength. It is known that there are technical difficulties such as that the surface roughness and the consistency of the film must be improved in order to prevent deterioration of characteristics due to scattering at the boundary surface of the film. There is.

[0011]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a DNA base sequencer capable of cutting off excitation light by means other than a filter.

[0012]

In order to solve the above-mentioned problems, according to the present invention, a vertically-held flat plate gel electrophoresis means having a large number of migration paths for DNA fragments, and the electrophoresis of the electrophoresis apparatus are provided. Laser light irradiation means for photoexcitation, which irradiates a laser beam so that the path is orthogonal to the migration path from the lateral direction of the electrophoretic device, and DNA irradiated by the laser light.
A DNA base sequence determination device comprising fluorescence detection means for detecting fluorescence generated from fragments and converting it into an electric signal, wherein a polarizing plate is provided between the fluorescence detection means and the electrophoretic plate. Provided is a DNA base sequencer. The polarizing plate is a polarizing plate that blocks the S-polarized component.

[0013]

The scattered light from the gel, which is a scatterer, preserves the polarization property of the excitation light (linear polarization), and the fluorescence property of the fluorescent substance is eliminated. In the present invention, paying attention to this characteristic, by arranging the P polarizing plate in front of the fluorescence detecting means, it has succeeded in cutting the excitation light and transmitting only the fluorescence.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in more detail below with reference to the drawings.

FIG. 1 is a partial schematic perspective view showing a schematic configuration of the DNA base sequence determination apparatus of the present invention. The fluorescence detecting means itself is not particularly limited. Any fluorescence detecting means conventionally used for fluorescence detection in an optical DNA nucleotide sequencer or fluorescence detecting means having a novel configuration other than that can be used.

The fluorescence detecting means shown in FIG. 1 is composed of a condenser lens 10 and a light receiving element 12, similar to the fluorescence detecting means shown in FIG. The polarizing plate 14 is arranged between the condenser lens 10 and the migration plate 74. Needless to say, the fluorescence detecting means can include the polarizing plate 14 as one of the components. Therefore, the polarizing plate 1
The arrangement position of 4 is not limited to the illustrated position. It may be between the lens 10 and the light receiving element 12. Further, it can be used together with a conventional wavelength cut filter.

As described above, the laser beam used in the DNA sequencer of the present invention is a linearly polarized laser having a wavelength of 488 nm. When this laser light passes through the gel electrolyte layer, the scattered light from the gel preserves the polarization characteristics of the excitation light. That is, the scattered light is linearly polarized light. On the other hand, the fluorescence from the fluorescent substance has a changed polarization direction and contains a large amount of P-polarized component. That is, the polarization characteristics of the fluorescence from the phosphor are eliminated.

Therefore, as shown in FIG. 2, when a polarizing plate that blocks the S-polarized light component and transmits only the P-polarized light component is used, the gel scattered light of the wavelength of 488 nm by the linear polarization laser for excitation of the wavelength of 488 nm is polarized. Blocked by a plate,
Only fluorescence with a wavelength of 520 nm is transmitted.

On the other hand, as shown in FIG. 3, when a polarizing plate that transmits the S-polarized component is used, both the gel scattered light and the fluorescent light are transmitted and the intended purpose cannot be achieved. Therefore, in the present invention, a polarizing plate that blocks the S-polarized component must be used.

The polarizing plate itself is known to those skilled in the art and will not require any particular explanation. Therefore, all conventionally used polarizing plates can be used in the device of the present invention. For example, a plastic polarizing plate or the like can be preferably used.

[0021]

As described above, according to the present invention,
By using the polarizing plate, only the fluorescence can be effectively and reliably transmitted to the fluorescence detecting means even if the wavelengths of the excitation light and the fluorescence are close to each other.

[Brief description of drawings]

FIG. 1 is a partial schematic perspective view showing a schematic configuration of a DNA base sequence determination device of the present invention.

FIG. 2 is a schematic diagram of a polarizing plate that detects a P-polarized component.

FIG. 3 is a schematic diagram of a polarizing plate that detects an S-polarized component.

FIG. 4 D disclosed in JP-A-63-21556
It is a typical block diagram of a NA base sequence determination apparatus.

FIG. 5 is a schematic configuration diagram of an example of a DNA base sequence determination device in which a fluorescence detection unit including a filter, a condenser lens, and a light receiving element is arranged for each migration path.

FIG. 6 is a characteristic diagram showing the relationship between the intensity and wavelength of excitation light with a wavelength of 488 nm and fluorescence with a wavelength of 520 nm.

[Explanation of symbols]

 10 Condensing lens 12 Light receiving element 14 Polarizing plate

Claims (3)

[Claims] 1. Having multiple migration paths for DNA fragments,
Plate-type gel electrophoretic means held vertically, and laser light irradiation means for photoexcitation for irradiating laser light on the electrophoretic path of the electrophoretic device so as to be orthogonal to the electrophoretic path from the lateral direction of the electrophoretic device. And a fluorescence detecting means for detecting fluorescence generated from the DNA fragment irradiated with the laser beam and converting it into an electric signal, wherein the fluorescence detecting means has a polarizing plate.
NA base sequencer. 2. The DNA base sequence determination device according to claim 1, wherein the polarizing plate is a polarizing plate that blocks S-polarized light components. 3. The laser light source is an argon ion laser which generates linearly polarized laser light having a wavelength of 488 nm, and D
FITC is used as a fluorescent substance for labeling the NA fragment, and when the fluorescent substance is irradiated with the laser light, fluorescence having a wavelength of about 520 nm is emitted from the FITC.
NA base sequencer.