JP2004191231A - Electrophoresis method and device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To realize an electrophoresis method and its device, being simplified, miniaturized and improved in its reliability and speed. <P>SOLUTION: A sample in a gel layer is electrophoresed by applying the voltage to upper and lower electrodes insulated from the gel layer while holding the gel layer therebetween. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an electrophoresis method and an electrophoresis apparatus used in the field of biotechnology and the like, and more particularly, to an improvement for reducing the size and speed of electrophoresis.
[0002]
[Prior art]
2. Description of the Related Art Conventionally, electrophoresis is 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]
Examples of electrophoresis include disk electrophoresis using polyacrylamide, SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis, isoelectric focusing, gel electrophoresis of nucleic acids, There are an electrophoresis method using the influence of interaction, a two-dimensional electrophoresis method, a capillary electrophoresis method, and the like.
[0004]
FIG. 4 is a configuration diagram showing an example of a conventional electrophoresis measurement device (see Patent Document 1). The apparatus is roughly divided into an electrophoresis device section 10 and a signal processing device section 20.
[0005]
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 the fluorescent substance, an optical fiber 17 for guiding light from the light source 16, and a fluorescent substance generated from the fluorescent substance. It comprises a photodetector 18 that collects fluorescence, selectively passes light of a specific wavelength by an optical filter, and converts the light into an electric signal.
[0006]
The signal processing unit 20 receives the electric signal from the photodetector 18 and can perform appropriate processing, for example, pre-processing such as data conversion into digital data and averaging processing. . 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.
[0007]
In such an apparatus, a gel is injected into the electrophoresis section 11, a sample of a DNA fragment labeled with a fluorescent substance is injected from above the gel, and then a voltage is applied to the first electrode 12 and the second electrode 13 by the power supply 15. Is applied, electrophoresis starts. In each lane of the sample, 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.
[0008]
For detection of these bands, the gel is irradiated with light (for example, laser light) from the light source 16 to generate fluorescence in the labeled fluorescent substance collected in the band in the gel, and this fluorescence is detected by the photodetector 18. To detect.
[0009]
That is, when the laser light is applied, the fluorescent substance of the gel existing on the optical path 31 is excited as shown in FIG. 5 to emit fluorescence. This fluorescence is detected at a predetermined detection position for each lane in the electrophoresis direction over time. Thus, fluorescence is detected when the band 32 of each lane passes through the position on the optical path 31, and 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.
[0010]
[Patent Document 1]
Japanese Patent Application Laid-Open No. 2000-338087 (page 2, FIG. 9, FIG. 10)
[0011]
[Problems to be solved by the invention]
However, such a conventional electrophoresis apparatus has the following problems. (1) Measurement takes time.
(2) A large number of lanes are required for various types of separation, which is large.
(3) The installation area is large. The area of the electrophoresis section is as large as 50 cm × 50 cm or 5 cm × 5 cm, for example.
[0012]
SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems and to realize a simple, compact, highly reliable, and high-speed electrophoretic method and an electrophoretic apparatus.
[0013]
[Means for Solving the Problems]
In order to achieve such an object, the invention of claim 1
A voltage is applied to upper and lower electrodes that are insulated from the gel layer with the gel layer interposed therebetween, thereby causing a sample in the gel layer to migrate.
[0014]
According to such a method, since the sample is forcibly drawn to the reactant side over a very short distance, there is an effect that the migration time can be significantly reduced as compared with the conventional method.
[0015]
In this case, a reactant that causes a chemical reaction and develops a color when the sample comes into contact with the gel layer is disposed on the gel layer, and the sample is drawn toward the reactant by the voltage application.
The voltage applied to the electrode is a DC voltage or a pulse voltage.
[0016]
Further, as in claim 4, the sample is dropped at a plurality of positions on the front surface side of the gel layer, and the reactant is disposed on the back surface side of the gel layer opposite to each sample. Thereby, a plurality of samples can be simultaneously migrated.
The electrode is insulated from the gel layer.
[0017]
The invention of claim 6 is
A film-like gel layer,
A sample dropped on the surface side of the gel layer,
A reactant that is disposed on the back side of the gel layer so as to face the sample, causes a chemical reaction when the sample comes into contact, and develops a color,
Electrodes respectively arranged on the front and back sides of the gel layer,
A power supply for applying a voltage to the electrode is provided, and by applying a voltage to the electrode, the sample migrates on the gel layer and is attracted to the reactant side.
[0018]
According to such a configuration, a simple and compact electrophoresis apparatus can be easily realized.
In addition, the portion composed of the gel layer, the sample, and the reactant can be removed from the electrode and can be handled as a disposable cartridge.
[0019]
The voltage applied to the electrode is a DC voltage or a pulse voltage.
Further, as in claim 8, the sample may be dropped at a plurality of locations on the front surface side of the gel layer, and the reactant may be arranged on the back surface side of the gel layer opposite to each sample. it can. Thereby, the samples are arranged in an array, and simultaneous migration of a plurality of samples becomes possible.
[0020]
In addition, the thickness and the material of the gel layer are different for each site where each sample is dropped, as in claim 9, and each site where each sample is dropped as in claim 10. It is also possible to provide a configuration in which individual electrodes are provided for each of the electrodes, and different voltages are applied to these electrodes.
[0021]
Further, in this invention, as in claim 11, the sample is negatively charged, the electrode disposed on the reactant side on the back surface of the gel layer is a positive electrode, and the electrode disposed on the gel layer front surface is Negative electrode. Further, the electrode is insulated from the gel. Furthermore, a light-absorbing reagent, a luminescent reagent or a fluorescent reagent is used as a reactant.
[0022]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail with reference to the drawings. The present invention is based on the principle of moving charged particles in a gel by applying an electric field to the charged particles. FIG. 1 is a main part configuration diagram showing an embodiment of the electrophoresis apparatus of the present invention.
[0023]
In FIG. 1, 1 is a gel layer, 2 is a sample, 3 is a reactant, 4 is a first insulating material, 5 is a second insulating material, 6 is a negative electrode, 7 is a positive electrode, and 8 is a power supply.
The gel layer 1 is formed of a thin film, for example, a film of about 1 μm or less. The sample 2 is, for example, target DNA and is dropped on the surface side of the gel layer 1. The reactant 3 is disposed on the back side of the gel layer 1 so as to face each sample 2, and has a property of causing a chemical reaction upon contact with the target DNA 2 to develop color.
[0024]
The insulating materials 4 and 5 are, for example, glass substrates and the like, which are arranged with the gel layer 1 interposed therebetween.
The insulating materials 4 and 5 form a part of a container (not shown), and the container is filled with the gel layer 1, the reactant 3, and the target DNA 2.
The negative electrode 6 is arranged above the first insulating material 4, and the positive electrode 7 is arranged below the second insulating material 5. A voltage is applied to this electrode from a power supply 8.
[0025]
In such a configuration, an appropriate high voltage in the range of, for example, several volts to several thousand volts is applied from the power supply 8 to the electrodes 6 and 7. When a high voltage is applied, an electric field is generated between the electrodes, and the negatively charged target DNA 2 is attracted to the positive electrode 7 side. When it passes through the gel layer 1 and comes into contact with the reactant 3, a chemical reaction occurs to develop color.
[0026]
If there is a difference in molecular weight between the respective target DNAs dropped on the gel layer 1, the distance traveled in the gel layer within the same time will be different. The lighter the molecule, the faster the migration speed, so that it reaches the reactant 3 earlier and develops a color.
By detecting the color development, it is possible to compare the molecular weights of DNA and determine the nucleotide sequence.
The color detection, the comparison of the molecular weight, and the determination of the base sequence are performed by well-known means (not shown).
[0027]
As described above, according to the present invention, since an electrochemical reaction that depends on the material of the electrode or the like does not occur, a highly reliable, simple, and compact electrophoresis apparatus can be easily realized.
In addition, since the present invention is not electrophoresis in which a current is applied as in the related art, electrolysis does not occur in the target DNA. Since the target DNA is attracted to the positive electrode side 7 with a large force by applying a high voltage, and is moved in the thickness direction of the thin film, the migration time is greatly reduced as compared with the conventional case.
[0028]
The present invention is not limited to the above-described embodiment, but includes many more changes and modifications without departing from the essence thereof.
For example, an electrophoretic array (also referred to as an electrophoretic array) can be formed by arranging the target DNA spots in an array and arranging them like a DNA chip.
[0029]
In addition, for each site where DNA is spotted, if the thickness and material of the gel layer are appropriately changed, or if individual electrodes are provided for each site and the applied voltage is changed for each site, electrophoresis can be performed in a two-dimensional array. it can.
In this case, if a transparent electrode such as ITO (indium-tin oxide) is used as the electrode, light emission, light absorption, fluorescence, and the like can be directly observed without removing the electrode.
[0030]
When the movement of the charged particles (DNA) is weak in the application of the DC voltage, a pulse voltage as shown in FIG. 2 may be applied. Since the structure of the embodiment is a capacitor structure, when a pulse is applied, a current flows at the moment of application. Therefore, based on the principle of electrophoresis, the target DNA 2 moves to the positive electrode 7 every time a pulse is applied as shown by an arrow in FIG.
[0031]
Further, a material having a high dielectric constant, for example, glycerin may be filled between the insulating material and the electrode to avoid an air gap.
In the embodiment, the insulating material 5 is inserted between the electrode and the gel layer. However, a liquid contact type without using the insulating material 5 may be used. In this case, the speed is increased by electrophoresis in the thickness direction of the gel layer. Can be realized.
[0032]
【The invention's effect】
As described above, the present invention has the following effects.
(1) An electrophoresis array can be easily formed by arranging sample drops on the gel layer in an array.
At this time, two-dimensional high-speed electrophoresis of the array can be easily performed at each site by changing the thickness or material of the gel layer or changing the applied voltage for each site.
(2) In the type having an insulator, a liquid contact electrode required in conventional electrophoresis is unnecessary, and a simple, compact, highly reliable electrophoresis method and electrophoresis apparatus can be easily realized.
(3) At this time, since electrolysis does not occur in the sample, a high voltage can be applied, and the speed of migration of the sample can be further increased.
[Brief description of the drawings]
FIG. 1 is a main part configuration diagram showing one embodiment of an electrophoresis apparatus according to the present invention.
FIG. 2 is an explanatory diagram of a pulse voltage waveform to be applied.
FIG. 3 is an explanatory diagram showing migration of a target DNA every time a pulse voltage is applied.
FIG. 4 is a configuration diagram illustrating an example of a conventional electrophoresis apparatus.
FIG. 5 is an explanatory diagram of an electrophoresis pattern.
[Explanation of symbols]
1 gel layer 2 sample 3 reactant 4,5 insulating material 6,7 electrode 8 power supply

Claims (13)

An electrophoresis method in which a voltage is applied to upper and lower electrodes sandwiching a gel layer in the thickness direction to cause a sample in the gel layer to migrate in the gel thickness direction. 2. The electrophoresis method according to claim 1, wherein a reactant that causes a chemical reaction and develops a color when the sample comes into contact is arranged on the gel layer, and the sample is drawn toward the reactant by the voltage application. 3. The electrophoresis method according to claim 1, wherein the voltage applied to the electrode is a DC voltage or a pulse voltage. 4. The method according to claim 1, wherein the sample is dropped at a plurality of positions on the front surface side of the gel layer, and the reactant is disposed on the back surface side of the gel layer opposite to each sample. Electrophoresis method as described. The electrophoresis method according to claim 1, wherein the electrode is insulated from the gel layer. A film-like gel layer,
A sample dropped on the surface side of the gel layer,
A reactant that is disposed on the back side of the gel layer so as to face the sample, causes a chemical reaction when the sample comes into contact, and develops a color,
An insulating material disposed on each of the front and back sides of the gel layer,
Electrodes respectively arranged outside the insulating material,
An electrophoresis apparatus, comprising: a power supply for applying a voltage to the electrodes; and applying a voltage to the electrodes so that the sample migrates on the gel layer and is attracted to the reactant. The electrophoretic device according to claim 6, wherein the voltage applied to the electrode is a DC voltage or a pulse voltage. 8. The electrophoresis device according to claim 6, wherein the sample is dropped at a plurality of positions on the front surface side of the gel layer, and the reactant is disposed on the back surface side of the gel layer opposite to each sample. apparatus. 9. The electrophoresis apparatus according to claim 6, wherein the thickness and the material of the gel layer are different for each site where each of the samples is dropped. 10. The electrophoresis apparatus according to claim 6, wherein an individual electrode is provided for each site where each of the samples is dropped, and different voltages are applied to these electrodes. 7. The method according to claim 6, wherein the sample is negatively charged, the electrode disposed on the reactant side on the back surface of the gel layer is a positive electrode, and the electrode disposed on the surface side of the gel layer is a negative electrode. 11. The electrophoresis apparatus according to any one of items 1 to 10. 12. The electrophoresis apparatus according to claim 6, wherein the electrode is insulated from the gel layer. 13. The electrophoresis apparatus according to claim 6, wherein the reactant is a light absorbing reagent, a luminescent reagent, or a fluorescent reagent.

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