JPH1123532A - Fluorescence detecting device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect fluorescence on a real-time basis and increase efficiency by arranging BPFs with different transmission regions in the plane perpendicular to the optical axis, spectrally splitting a parallel light beam in different areas, and converging the beams on separate sensors with separate lenses. SOLUTION: When excitation light is fed to a fluorescence marker sample migratingly arrived along a gel electrolyte layer in a glass tube 1, fluorescence is generated. The fluorescence is received by a condensing lens 9 set with its focal position at the center of the glass tube 1 as parallel beam. Four kinds of BPF(BP1 -BP4 ) transmitting the vicinities of the peak wavelengths of fluorescence dyes are arranged on the plane perpendicular to the optical axis in the optical path of a detection system. The received parallel beam is spectrally split in four areas. Light is converged on four split sensors 15 by collective lenses 13 in the sensor areas S1 -S4 of the sensor 15 in the corresponding relation of BP1 :S1 -BP4 :S4 . The sensor having the maximum sensor output in the sensor areas S1 -S4 is judged, the dye of the corresponding fluorescence peak wavelength is determined, and the color is discriminated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] 1. Field of the Invention [0002] The present invention relates to a multicolor fluorescence detecting device. More specifically, the present invention relates to a gel electrophoresis apparatus for electrophoresing DNA constituent bases labeled with a plurality of fluorescent dyes.

[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 DNA fragment labeled with a fluorescent dye is electrophoresed in a gel, and a photoexcitation section and a photodetector are provided for each electrophoresis path 15 to 20 cm below the electrophoresis start section. The DNA fragments passing here are measured in order. For example, DNAs of various lengths having no terminal base species 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, an adenine (A) fragment group, a cytosine (C) fragment group, a guanine (G) fragment group, and a thymine (T) fragment group labeled with a fluorescent substance are obtained. These fragment groups are mixed, injected into separate electrophoresis lane grooves of an electrophoresis gel, and a voltage is applied. 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.

[0005] Dyes currently used for labeling DNA in the determination of base sequence include, for example, fluorescein isothiocyanate (FITC), eosin isothiocyanate (EITC), tetramethyl rhodamine isothiocyanate (TMRITC) and rhodamine substituted isothiocyanate. (XRITC). Instead of labeling the specimen with one type of dye, labeling with two types of dyes and measuring two types of fluorescence improve analysis throughput.

For example, when FITC and XRITC are used as fluorescent dyes for labeling DNA fragments,
When this fluorescent dye is irradiated with laser light, it emits fluorescence with wavelengths of about 520 nm and 604 nm, respectively. When two types of fluorescent dyes are used, the number of samples per gel can be simply doubled as compared with the case where one type of fluorescent dye is used, and the discriminating base length can be increased. For example, when one type of fluorescent dye is used, the number of lanes per sample is four, and the number of fluorescent dyes is two.
This is the same as the case of using one kind, but the number of samples per gel is 8 when one kind of fluorescent dye is used, whereas the number of samples is doubled to 16 when two kinds of fluorescent dyes are used.
When one type of fluorescent dye is used, the discriminating base length is 4
In contrast to 00 base pairs, when there are two types of fluorescent dyes,
The discriminating base length can be increased to 450 base pairs.

In recent years, in order to further improve the throughput of analysis, four bases of DNA have different peak wavelengths.
A multicolor DNA sequencer using a sample labeled with a single fluorescent dye has been developed. In such a conventional multicolor DNA sequencer, for example,
A method is adopted in which a plurality of bandpass filters are arranged on a disk, and the disks are sequentially switched by rotating the disk in a detection optical path to detect fluorescence of a sample. However, in this method, it is necessary to switch the filter over time, so that real-time detection is not possible, and a rotating mechanism is required. Become As another method, a method is adopted in which a spectral prism is inserted in the middle of the fluorescence detection optical system to perform spectral analysis of a sample. However, in this method, the light captured by the condenser lens is converted into parallel light, and after being separated by the prism, each color is collected on the sensor, but it is necessary to correct chromatic aberration, aberration by the prism, and the like.
There is structural difficulty.

[0008]

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a highly efficient and compact multi-color fluorescence detecting device capable of real-time detection.

[0009]

The object of the present invention is to dispose a band-pass filter having different transmission areas in a plane perpendicular to the optical axis to separate parallel light beams into areas, and to further separate the light split into light in each area. This is solved by focusing the light on separate sensors, each with a separate lens.

[0010]

FIG. 1 is a schematic perspective view showing the structure of an example of a multicolor fluorescence detecting device according to the present invention. In the multicolor fluorescence detection device of the present invention, the glass tube 1 (inner diameter <
A 100 μm) polyacrylamide gel or other suitable polymer as an electrophoresis medium is filled, and a sample in which each base constituting DNA is labeled with four fluorescent dyes is placed in the same glass tube and subjected to electrophoresis.

A predetermined length of the glass tube 1 (for example, 30
A laser for fluorescence excitation is positioned sufficiently small (for example, 0.1 cm) at the position where electrophoresis has been performed.
〜0.4 mm). The irradiation system includes a laser light source 3, a collimator lens 5, and a cylindrical lens 7.
Consists of The type of the laser light source 3 is not particularly limited,
Due to the relationship with the fluorescent dye used, for example, when the excitation wavelength is 4
It is preferable to use an 88 or 514.5 nm argon laser or the like.

When the excitation light from the irradiation system is incident on the fluorescently labeled sample migrated along the gel electrolyte layer in the glass tube 1, fluorescence is generated from this sample. The generated fluorescence is taken into the detection system as parallel light by the condenser lens 9 whose center is set at the focal position of the glass tube 1. In the optical path of the detection system, four types of band-pass filters (Bp _{1 to} Bp ₄ ) 11 transmitting near the peak wavelength of each fluorescent dye 11
a, 11b, 11c and 11d are integrally arranged in a plane perpendicular to the optical axis. The captured parallel light is divided by the filters 11a to 11d into four areas in a plane perpendicular to the optical axis, and is separated. The split light is a collective lens 13a, 13b, 13 provided for each area.
According to c and 13d, Bp ₁ : S ₁ , B ₁ for each sensor area (S _{1 to} S ₄ ) of the four-divided sensor 15.
Light is condensed on the sensor 15 in a correspondence relationship of p ₂ : S ₂ , Bp ₃ : S ₃ and Bp ₄ : S ₄ . The four-divided sensor 15 is, for example, 4
Split photodiode sensors can be used.

The color, that is, the determination of the base is determined, for example, by S ₁
Of the sensor output up to S _4, by determining the sensor to be a maximum, is performed by the corresponding fluorescent peak wavelength dye is determined. Alternatively, the amount of individual dye leaking into each filter may be determined, and the dye may be determined by performing an inverse calculation on the detected signal.

FIG. 2 is a characteristic diagram showing the relationship between the characteristics of each fluorescent dye and the transmission region of the band-pass filter. The vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the wavelength. In FIG. 2, F
L _{1 to} FL ₄ are waveforms indicating the fluorescence spectral characteristics of each fluorescent dye, and Bp _{1 to} Bp ₄ indicate the transmission region of the band-pass filter. As shown, each bandpass filter can specifically transmit the peak wavelength of each fluorescent dye, and has excellent separation characteristics.

Although only one glass tube 1 is shown in FIG. 1, a plurality of (for example, 40 to 100) glass tubes can be used. In FIG. 1, the irradiation system and the detection system are arranged so as to be orthogonal to each other across the glass tube.
The arrangement is not limited to this arrangement. For example, the irradiation system and the detection system may be arranged so as to face each other with the glass tube interposed therebetween. Although not shown, the upper part of the glass tube is immersed in the buffer solution of the upper buffer tank, and the lower part is immersed in the buffer solution of the lower buffer tank. An upper electrode is further provided in the upper buffer tank, and a lower electrode is provided in the lower buffer tank. Electrophoresis is performed by applying a voltage between the electrodes. A gel electrophoresis apparatus using such a plurality of glass tubes is disclosed in, for example, JP-A-5-72177.

[0016]

As described above, according to the present invention,
Since each sensor can detect fluorescence in real time, sensitivity can be improved. Further, since the light is separated at the optical axis parallel to the optical axis, the light is efficiently separated. Further, since no driving system is required at all mechanically, it is possible to reduce the size of the overall apparatus.

[Brief description of the drawings]

FIG. 1 is a schematic perspective view showing a configuration of an example of a multicolor fluorescence detection device of the present invention.

FIG. 2 is a characteristic diagram showing a relationship between characteristics of each fluorescent dye and a transmission region of a bandpass filter.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Glass tube 3 Laser light source 5 Collimator lens 7 Cylindrical lens 9 Condensing lens 11 Bandpass filter 13 Collective lens 15 Four-split sensor

Claims (4)

[Claims] 1. A glass tube on which a sample labeled with four kinds of fluorescent dyes is electrophoresed, an irradiation system for irradiating the glass tube with excitation light, and detecting fluorescence emitted from the sample irradiated with the excitation light. In a fluorescence detection device including a detection system, the detection system includes a condenser lens for condensing fluorescence,
Kinds of bandpass filters, 4 collective lenses, 4
A fluorescence detection device comprising: a plurality of divided sensors. 2. A condenser lens is set at a focal position at the center of a glass tube, four types of band-pass filters are integrally disposed in a plane perpendicular to an optical axis, and four collective lenses are optical The apparatus of claim 1 wherein the bandpass filters are integrally disposed in a plane perpendicular to the axis, and each bandpass filter corresponds to a respective sensor on each optical axis. 3. The apparatus according to claim 1, wherein a plurality of glass tubes are used in a bundle. 4. The device according to claim 1, which is a gel electrophoresis device.