JPH05322770A - Multi-marker electrophoresis - Google Patents

Abstract

PURPOSE:To compare and identify a reference sample and a test sample with high precision by concurrently applying electrophoresis on the same gel. CONSTITUTION:Markers are given to a reference sample and a test sample with-fluorescent materials having different inherent excitation frequencies or fluorescence wavelengths or luminescent materials having different luminescence wavelengths. Electrophoresis is concurrently applied to the reference sample and the test sample on the same gel after both samples are mixed, the fluorescent material or the luminescent material marked with the reference sample and the test sample is detected from the obtained bands, and both samples are compared and identified. The light emitted from an argon laser 1 is bent by a mirror 2 and radiated to an electrophoresis plate 3, and a laser beam is scanned on the electrophoresis plate 3. Electrophoresis is made on the electrophoresis plate 3, and bands of the reference sample and the test sample separated by electrophoresis exist on the electrophoresis plate 3. The fluorescence emitted from individual fluorescent materials is condensed by a lens 4 and enters an interference filter unit 5. The unit 5 rotates the filters having different wavelengths with a motor. The fluorescence passing through the filter is fed to a photomultiplier 6 and detected.

Description

Detailed Description of the Invention

[0001]

The present invention relates to proteins, nucleic acids, DNA
The present invention relates to an electrophoretic method for performing rapid and accurate comparative identification such as.

[0002]

2. Description of the Related Art When a substance, such as a protein, having a positive or negative charge on its surface is placed under an electric field, it is separated due to a difference in mobility or the like due to a difference in charge magnitude or molecular weight of the substance. It In particular, gel electrophoresis, in which separation is performed in a support such as gel, has been performed since 1959 when polyacrylamide gel electrophoresis was performed, and gel disc electrophoresis, SDS
Many electrophoretic methods such as gel disc electrophoresis and isoelectric focusing have been developed and widely used for the purpose of separating and analyzing proteins and nucleic acids in genetic engineering.

As a detection method, when the sample is a protein,
The gel separated by electrophoresis is stained with Coomassie-Brilliant Bull and silver. When using DNA as a target, staining with ethidium bromide or detection with a radioisotope (RI) label is performed. In general, when the dyeing method is used, it takes a day and night for the dyeing step and the decolorizing step, the sensitivity is low (in the case of Coomassie-Brilliant Bull), and the problem is that it is a carcinogen (in the case of ethidium bromide). There is a point. In many cases, RI-labeled substances are electrophoresed because of their high sensitivity, but RI is not only a major safety issue, it is also cumbersome and time-consuming. When RI is used, it takes time to expose the film. Therefore, the use of a fluorescent substance as a label has been reported. DNA sequencer
The type of terminal base (adenine (A), guanine (G), thymine (T), cytosine is added to the DNA fragment that has been previously treated by the Sanger method.
(C)) with different fluorescent substances attached, run in a single migration lane, and are excited by laser light to receive light of each fluorescence wavelength Some electrophoretic patterns are detected.

[0004]

[Problems to be Solved by the Invention] By applying electrophoresis as described above, (1) determination of whether or not a mutation has occurred in DNA, (2) comparison of DNA of mouse or other type or pathological mouse , (3) of the gene-incorporated mouse
DNA comparisons are possible. These experiments are performed by comparative identification with an electrophoretic image (band) with a standard sample. In the electrophoresis method, the gel is electrophoresed in one direction, but conventionally, the standard sample and the sample sample were separately electrophoresed, and comparative identification was performed by the positions of the bands obtained from the standard sample and the sample sample, respectively. Was there. However, the gel preparations are not exactly the same, and even in the same gel, the position of the obtained migration band is often distorted due to the variation in the heat distribution during migration between each lane. , Causes measurement error.
Therefore, although measures such as cooling of the migration tank and distortion correction by image processing have been taken, even if samples of the same type are migrated, it is impossible to completely match the migration images. Further, the distortion correction apparatus by the image processing described above is very expensive and requires a lot of time for correction, and the obtained results also have the drawback that an image with completely distorted images cannot be obtained. Therefore, accurate comparative identification and analysis are impossible.

[0005]

In view of the above problems, an object of the present invention is to eliminate distortion correction by image processing,
It is to provide an electrophoresis method. That is, according to the present invention, a standard sample (for example, normal cell DNA) and a specimen sample (for example, cancer cell DNA) are labeled with fluorescent substances having different excitation wavelengths or fluorescence wavelengths, or luminescent substances having different emission wavelengths. (Hereinafter, the labeling substance for the standard sample is referred to as A and the labeling substance for the analyte substance as B), and after mixing both samples, the standard sample and the analyte sample are simultaneously subjected to electrophoresis on the same gel to obtain From the band
This is an electrophoretic method characterized by comparing and identifying a standard sample and an analyte sample by detecting respective signals of B. In the present invention, the standard sample and the sample sample are described in the case of one sample, but each sample may be a plurality of samples. In the electrophoresis method according to the present invention, even if the standard substance and the sample substance are developed at the same position, the excitation wavelength of the standard substance, or the fluorescence wavelength, or the emission wavelength is different depending on the standard sample and the sample sample. It is possible to detect the standard substance applied to the sample, and if a signal of only A is obtained from a certain band, it was found that the sample sample does not contain the molecular weight substance. , If both signals are obtained, it is possible to identify the molecular weight substance of the specimen sample and the standard sample, and if only the signal of B is obtained, it is found that the specimen sample has a molecular weight substance which does not exist in the standard substance. To do. In the electrophoretic method according to the present invention, both samples (standard sample and specimen sample) are simultaneously electrophoresed on the same gel and the same lane, so that they are affected by the distortion of the spot due to the gel adjustment. Not ideal electrophoresis can be constructed. Use of the present invention facilitates analysis of genetic abnormalities,
Moreover, since it can be carried out with high accuracy, research on genetic abnormalities can be dramatically advanced.

As the labeling substance, a fluorescent substance, a luminescent substance or the like is used. When fluorescent substances are used, labeling substances A and B
Must use substances with different excitation or fluorescence wavelengths. As an example, Table 1 shows combinations of fluorescent substances,
Here are some: (In Table 1, λex represents the excitation wavelength and λem represents the fluorescence wavelength.) In Table 1, the excitation light side is the same in (1) and (2) (excitation wavelength near 495 nm in (1), (2 In (), a light source or (light source + interference filter) with an excitation wavelength of 325 nm is used, and interference filters for the fluorescence wavelengths of A and B are provided on the fluorescence receiving side, and conversely in (3) and (4). Are the same (in (3), an interference filter around 520 nm, and in (4) an interference filter around 578 nm is installed in front of the light receiving element) Then, each interference filter is alternately replaced and the signal is taken in synchronism with it.In order to improve the S / N, one having a narrow half width of the interference filter is selected, or A, B is selected.
A combination having a large difference in excitation wavelength, fluorescence wavelength, or emission wavelength may be selected. In the method of the present invention, each of the standard substances A and B is not limited to one type, but by selecting an interference filter having a small full width at half maximum, that is, measuring each wavelength separately. Thus, by selecting two or more types of labeling substances A and B for two or more types of standard samples and specimen samples, respectively, electrophoresis can be performed simultaneously on the same gel and each sample can be compared and identified. Hereinafter, the method of the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these disclosures.

[Table 1]

[0007]

[Embodiment 1] In FIG. 1, the light emitted from the argon laser-1 is bent by the mirror-2 and illuminates the migration plate. The mirror is moved by the motor so that laser light scans the electrophoresis plate. The electrophoresis plate is a plate on which electrophoresis has already been performed and bands of the standard sample and the analyte sample separated by electrophoresis are present on the plate.

Normal rat (hereinafter referred to as X) as a standard sample and Rhodamin sarcoma as a specimen sample were transplanted 2
Serum obtained from a week-old rat (hereinafter referred to as Y) was used. The protein in the serum from X was labeled with merocyanine 540, and the protein in the serum from Y was labeled with FITC by known methods. The electrophoresis was performed by polyacrylamide gel. The gel concentration was 10% to 20% linear acrylamide gradient gel, and 20 mA (17 hours) was applied. As the migration buffer, 0.025 mol Tris-0.192 mol glycine was used. The fluorescence generated from each fluorescent substance is condensed by the lens 4 and the interference filter-unit 5
Can be entered. Interference filter-unit 5 is 570nm
The 520 nm interference filter holder is equipped with a mechanism that rotates by a motor, so that the labeled fluorescent substance merocyanine 540 and FITC can be alternately detected. The fluorescence passing through the interference filter unit enters the photomultiplier tube 6 which is a detector and is detected. This signal is processed in synchronism with the switching of the interference filter--the filter of the unit--to determine whether it is from serum from X or serum from Y. It was possible to compare two signals of the respective labeled substances obtained at the same time from the same gel, confirm the migration position of the specimen sample of the standard sample on the gel, and compare and identify the two.

[0009]

Example 2 Standard and specimen samples are treated with a 6-base recognition restriction enzyme. After reacting the standard sample with merocyanine 540-labeled nucleotide and the sample sample with FITC-labeled nucleotide, both samples were mixed and electrophoresed.
3 is a vertical slab gel electrophoresis plate using 8% polyacrylamide gel containing 7 mol of urea. TBE buffer was used as the buffer.

Fluorescence generated from each fluorescent substance is condensed by the lens 4 and is input to the interference filter unit 5. The interference filter unit 5 is provided with a mechanism in which an interference filter holder of 570 nm and 520 nm is rotated by a motor, and is a labeling fluorescent substance, merocyanine 540, FITC.
Can be detected alternately. Interference filter
The fluorescence passing through the unit enters the photomultiplier tube 6 which is a detector and is detected. This signal is processed in synchronism with the switching of the interference filter--the filter of the unit--to determine whether it is from the standard sample DNA fragment or the sample DNA fragment. In this way, the two signals for the respective labeled substances obtained from the same gel were compared, the migration position of the specimen sample for the standard sample on the gel was confirmed, and the comparative identification of both was possible. .. Since the difference in the migration position between the standard sample and the specimen sample is caused by the difference in the DNA base sequence of the gene, the gene abnormality could be analyzed by detecting this difference.

[0011]

In the electrophoretic method of the present invention, the standard sample and the specimen sample are electrophoresed on the same gel at the same time, so that they can be compared with high accuracy without being affected by the distortion of the electrophoretic spot position. Identification is possible.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a method for detecting a labeling substance from the electrophoretic gel (electrophoresis plate) obtained by the method of the present invention shown in Examples 1 and 2. FIG.

[Explanation of symbols]

 1. Laser oscillator 2. Reflection mirror-3. Electrophoresis plate 4. Lens 5. Interference filter-unit 6. Photomultiplier tube

Claims (1)

[Claims] 1. When performing electrophoresis, a standard sample and an analyte sample are labeled with fluorescent substances having different unique excitation wavelengths or fluorescent wavelengths or luminescent substances having different emission wavelengths, and both samples are mixed. After that, the standard sample and the sample sample are simultaneously electrophoresed on the same gel, and the fluorescent substances or luminescent substances labeled on the standard sample and the sample sample are detected from the obtained band to compare and identify the standard sample and the sample sample. A multi-label electrophoresis method characterized by being performed.