JPH10176992A - Dna-base-array decision apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a DNA-base-array decision apparatus in which the influ ence of background light can be reduced to a minimum when fluorescence is detected by a fare irradiation system. SOLUTION: In a DNA-base-array decision system provided with an irradiation system and a light-receiving system, a DNA fragment sample 76 which is labeled by a phosphor in a gel electrolyte layer 78 is excited through a glass migration plate 74 from the surface or the rear of the glass migration plate 74, and fluorescence generated at this time is detected from the surface or the rear of the migration plate 74. In the DNA-base-array decision apparatus, the irradiation system is arranged and installed with reference to a glass face at an angle of the Brewster angle decided by the refractive index of a glass used for the migration plate 74.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a gel electrophoresis apparatus. More specifically, the present invention relates to a base sequence determination apparatus for labeling a DNA fragment sample with a fluorescent substance, performing gel electrophoresis, and determining the base sequence thereof.

[0002]

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

Conventionally, when performing electrophoresis, a sample is labeled with a radioisotope and analyzed, but this method has a drawback that it requires time and effort. In addition, maximum safety and control are always required from the viewpoint of radioactivity management, and analysis cannot be performed unless 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, a fluorescently labeled DN is used.
The A fragment is allowed to migrate in the gel.
A photoexciting section and a photodetector are provided for each electrophoresis path below ２０20 cm, and DNA fragments passing therethrough are sequentially measured. For example, DNAs of various lengths whose terminal base species are known are duplicated by an enzymatic reaction (dideoxy method) using a DNA chain whose sequence is to be determined as a template, and these are labeled with a fluorescent substance. 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 fragment groups are mixed and injected into separate electrophoresis lane grooves of an electrophoresis gel,
Apply voltage. Since DNA is a chain polymer polymer having a negative charge, it moves in the gel at a speed inversely proportional to the molecular weight. Shorter (small molecular weight) DNA strands move faster and longer (larger molecular weight) DNA chains move more slowly, so that DNA can be fractionated by molecular weight.

FIG. 7 is a schematic perspective view of an example of an apparatus for determining a DNA base sequence by gel electrophoresis using a fluorescent label. In this apparatus, the DNA fragment sample 76 is irradiated with laser light 90 from the front surface of the electrophoresis plate 74 by the light source 70, and the generated fluorescence is received on the same surface side. The fluorescent light is condensed by the condensing lens 102, and further, the excitation light
4 and detected by a fluorescence detector 106 (eg, a CCD or photomultiplier). The irradiation method as shown in FIG. 3 is generally called a surface irradiation method.

In the surface irradiation method, the excitation light is made incident from the surface of the electrophoresis plate (glass plate). Background light (stray light component) tends to be larger than fluorescence to be detected. When the background light (stray light component) increases, the dynamic range of the fluorescence detector is narrowed, which causes inconvenience when detecting the fluorescence.

[0007]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a DNA base sequencer capable of minimizing the influence of background light when performing fluorescence detection by a surface irradiation method.

[0008]

The object of the present invention is to excite a DNA fragment sample labeled with a fluorescent substance in a gel electrolyte layer from the front or back surface of a glass electrophoresis plate through the electrophoresis plate, and migrate the fluorescence generated at that time. In the DNA base sequencer having an irradiation system and a light receiving system, which is detected from the front surface or the back surface of the plate, the irradiation system has a Brewster angle determined from the refractive index of the glass used for the electrophoresis plate. The problem is solved by disposing it on the glass surface.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a schematic sectional view of one example of a DNA base sequence determination apparatus according to the present invention. As shown in the figure, the electrophoresis plate 74 is composed of two glass plates, and a gap between the glass plates is filled with a layer 78 made of a gel electrolyte such as polyacrylamide. DN labeled with phosphor
The A fragment sample 76 migrates from one end of the electrophoresis plate to the other end of the electrophoresis plate due to charges in the gel electrolyte layer 78.

A light receiving system comprising a condenser lens 1, an excitation light cut filter 3 and a fluorescence detector 5 is arranged perpendicular to the surface of the glass plate. On the other hand, the irradiation system including the light source 7 and the collimator lens 9 is arranged to be inclined by the angle θ. Therefore, this angle θ is the incident angle of the excitation light.

The magnitude of the background light detected by the light receiving system changes according to the incident angle of the irradiation light depending on the angle with respect to the glass surface of the light receiving system. The reflectance on the glass surface of the irradiation system varies depending on the polarization component, but the reflectance of the P component is 0% at Brewster's angle, and the magnitude of the background light is expected to show a minimum value at this angle. Is done. That is, it is considered that the best S / N ratio is obtained when the excitation light is irradiated at the Brewster angle. The “Brewster angle” is the angle of incidence at which the reflectivity of light whose electric vector is within the plane of incidence is zero with respect to light reflected on a dielectric surface (Shoji Koyanagi, “Optical Technical Glossary”, Published by Optronics in January 1994).

The irradiation system is preferably non-polarized. The incident angle θ of the irradiation system is a Brewster angle determined from the refractive index of the glass plate used. Therefore, the incident angle θ changes depending on the glass plate of the electrophoresis plate used. FIG. 2 is a characteristic diagram obtained by measuring the relationship between the magnitude of background light and the incident angle on a glass plate generally used as a migration plate in a gel electrophoresis apparatus. The magnitude of the background light was measured at three different locations on the glass plate. The magnitude of the fluorescence (F _I ) due to the surface irradiation is substantially constant regardless of the incident angle. On the other hand, the magnitude (I _BG ) of the scattering (background light) due to the surface irradiation becomes smallest when the incident angle is around 55 to 60 °.

FIG. 3 shows the magnitude of fluorescence (F _I ) due to surface irradiation.
FIG. 9 is a characteristic diagram showing a ratio of a magnitude (I _BG ) of scattering (background light) by surface irradiation with respect to. As is clear from this characteristic diagram, the ratio becomes almost maximum when the incident angle θ is around 55 to 60 °. Therefore, from these characteristic diagrams, in the case of this glass plate, the irradiation system is set to 55 to 6 with respect to the glass surface.
It can be seen that it should be arranged at an angle of 0 °.

The type of the light source 7 used in the irradiation system is not particularly limited. Any light source such as a laser light source, an incandescent light source, or a laser diode that can excite a fluorescent substance used to label a DNA fragment sample and generate fluorescence therefrom can be used. The same number of light sources as the number of migration paths can be used, or they can be commonly used for all migration paths by scanning with an appropriate drive system. Therefore, at least one light source is required. Other optical means such as the lens 9 used in combination with the light source may be appropriately selected and used as needed.

Similarly, the combination of the condenser lens 1, excitation light cut filter 3 and fluorescence detector 5 of the light receiving system is not limited to the configuration shown in FIG. For example, Japanese Patent Application No. 6-18
A selfoc lens or a CCD line sensor as disclosed in Japanese Patent No. 3028 can also be used.

As described above, the reflectance and the transmittance of the incident light on the glass surface depend on the polarization component (P component and S component) depending on the incident angle.
Component). P of the polarization component
Regarding the components, the reflectance can be made almost 0% by setting the incident angle to the Brewster angle. However, even if the incident angle is set to Brewster's angle, the S-polarized light component is not cut, but is incident on the fluorescence detector together with the P-polarized light component, and causes disturbance of the detector. Therefore, this S-polarized component is cut,
The best results are obtained if only the P-polarized component is incident on the fluorescence detector.

FIG. 4 is a partial schematic cross-sectional view of an example of the DNA base sequence determination apparatus of the present invention in which a polarizing plate 11 for cutting the S-polarized component incident on the fluorescence detector is inserted in the optical path of the light receiving system. FIG. 5 is an output waveform diagram of the fluorescence detector when a polarizing plate for cutting the S-polarized component is not used.
FIG. 6 is an output waveform diagram of the fluorescence detector when a polarizing plate for cutting the S-polarized component is used. By comparing both figures, it can be seen that the use of a polarizer keeps the background low and relatively widens the dynamic range of the detector. The polarizing plate 11 can be inserted not only in the optical path of the light receiving system as shown, but also in the optical path of the illumination system.

[0018]

As described above, according to the present invention,
Excitation light is incident on the glass surface at a Brewster angle determined from the refractive index of the glass used for the electrophoresis plate. As a result, the background output can be minimized, and the dynamic range of the fluorescent signal can be widened, and a good S / N ratio can be obtained.

[Brief description of the drawings]

FIG. 1 is a partial schematic cross-sectional view of an example of a DNA base sequencer according to the present invention.

FIG. 2 is a characteristic diagram showing a relationship between an incident angle of excitation light and a background output on a glass plate used as an electrophoresis plate.

FIG. 3 is a characteristic diagram showing a ratio between a background output and a fluorescence output shown in FIG. 2;

FIG. 4 is a partial schematic cross-sectional view of an example of the DNA base sequence determination apparatus of the present invention in which a polarizing plate for cutting an S-polarized component is inserted into a light receiving system optical path.

FIG. 5 is an output waveform diagram of a fluorescence detector when a polarizing plate for cutting an S-polarized light component is not used.

FIG. 6 is an output waveform diagram of a fluorescence detector when a polarizing plate for cutting an S-polarized light component is used.

FIG. 7 is a partial schematic cross-sectional view of an example of a conventional DNA base sequence determination device.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Condensing lens 3 Excitation light cut filter 5 Fluorescence detector 7 Light source 9 Collimator lens 11 S-polarization component cut polarizing plate 74 Electrophoretic plate 76 Fluorescent substance labeled DNA fragment sample 78 Gel electrolyte layer

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C12M 1/00 G01N 27/26 325A

Claims (3)

[Claims] 1. A DN labeled with a fluorescent substance in a gel electrolyte layer from the front or back of a glass electrophoresis plate through the electrophoresis plate.
In a DNA sequencer having an irradiation system and a light receiving system, which excites an A fragment sample and detects fluorescence generated at that time from the front or back surface of the electrophoresis plate, the irradiation system is a glass used for the electrophoresis plate. A DNA base sequence determining device disposed at an angle of Brewster's angle determined from the refractive index of the glass surface. 2. An irradiation system and a light receiving system are both arranged on the same surface side of one glass plate of the electrophoresis plate, the light receiving system is arranged perpendicular to the glass surface, and the irradiation system is arranged on the glass surface. 2. The DNA base sequencer according to claim 1, wherein the DNA base sequencer is arranged to be inclined at a Brewster angle determined from the refractive index of the glass. 3. The DNA base sequencer according to claim 1, further comprising a polarizing plate for cutting an S-polarized component.