JP2001133438A - Ultracompact thin layer gel electrophoresis apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an ultracompact electrophoresis apparatus having a high separation efficiency and a high detection sensitivity. SOLUTION: The ultracompact thin layer gel electrophoresis apparatus uses as a support a flat gel comprised of a concentration gel layer 5 of a thickness of at least 50-350 μm and a separation gel layer 6 of a thickness of not larger than 50 μm.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microminiature thin-layer gel electrophoresis apparatus. More specifically, the invention of this application relates to a microminiature thin-layer gel electrophoresis apparatus having high separation efficiency and high detection sensitivity.

[0002]

2. Description of the Related Art Electrophoresis is a separation and analysis method utilizing the phenomenon that, when a charged molecule is placed in an electric field, it moves in the direction of an electrode of opposite polarity. In a gas phase, molecules move in the direction of the electrode while accelerating because there is almost no resistance. In the electrophoresis method, a support such as an aqueous solution or a gel is generally used as a field for electrophoresis.

Conventionally, in the field of life science, gel electrophoresis has been widely used for analysis of biological macromolecules such as DNA and proteins. In the analysis of protein and DNA molecules, it is necessary to control the resistance to the movement of the molecules in order to improve the resolution, and gel electrophoresis using a gel as a support is known as a method for obtaining high resolution. Have been.
In gel electrophoresis, a network (gel) is formed by macromolecules and the size of the pores of the stitch is adjusted to create a sieving effect. Can be reflected. Further, when the solution is held in the gel, there is an advantage that the heat convection of the solution is suppressed by the network of the gel.

[0004] Gel electrophoresis is classified according to the driving force of electrophoresis, its control method, the shape of a support, and the like. The main purpose of the method of causing a difference in the movement speed depending on the polymer network is estimation of the molecular size. For this purpose, it is desirable that the sample molecules have a charge number proportional to the size. DNA molecules have a phosphate group as a dissociating group that affects charge, which gives one negative charge per base unit of DNA. Therefore, since the charge number is proportional to the length of the molecule, it is relatively easy to determine the size of the molecule by gel electrophoresis. On the other hand, in a protein molecule, since the PKa of the dissociating group providing a charge is in the range of about 3 to 12, the charge number has no relation to the molecular size as it is. Therefore, in the case of proteins, SDS gel electrophoresis in which sodium dodecyl sulfate (SDS), which is an anionic surfactant, is added and a dodecyl sulfate group (giving one negative charge per molecule) is used. . Since the amount of the dodecyl sulfate group is proportional to the molecular size of the protein, the charge number is approximately proportional to the molecular size, and the molecular size can be approximately determined from the speed of movement in the gel.

As another method for performing electrophoretic separation by controlling the driving force, there is an isoelectric focusing method. This is a method in which a pH gradient is formed in a support gel sandwiched between electrodes, and proteins are separated from each other by converging proteins at the positions of their unique isoelectric points. Hydrogen ions are supplied from the anode electrode solution, and hydroxide ions are supplied from the cathode electrode solution.When a DC voltage is applied between the electrodes, the pH supplied to the support is increased by the ions supplied from both electrodes. A gradient is formed. The inclusion of benign electrolyte mixtures with different isoelectric points allows them to be arranged in the support in the order of their isoelectric points by the application of a voltage, thus stabilizing the pH gradient. The protein molecule converges to its isoelectric point in the stabilized pH gradient. In isoelectric focusing, the support does not necessarily need to be a gel for separation, but a gel with a low density is used as the support to suppress thermal convection and diffusion of molecules.

[0006] The shape of the support in the gel electrophoresis method is roughly divided into a gel made of polyacrylamide or the like between a pair of opposing translucent plates, and both ends connected to an electrode tank containing an electrode solution. , Energization and separation of the mixture containing the target substance by flat gel electrophoresis, gel formation in capillaries, or dissolving hydrophilic linear polymers in water Capillary electrophoresis, in which a similar structure is formed, and a high voltage is applied between both ends to separate and analyze a mixed solution containing a target substance.

[0007] In any of the methods, after the target substance is separated, the target molecule is detected using spectroscopy or the like. In slab gel electrophoresis, gels are generally stained, the state of the staining is recorded using a single-lens reflex camera, video camera, or gel scanner, and separated by laser scanning, image processing, data analysis using software, etc. Analyze DNA and proteins. In capillary electrophoresis, the separated target molecule is detected by ultraviolet absorption or fluorescence analysis.

[0008] In the plate gel electrophoresis method, a large number of samples (usually 5 to 20) can be injected and separated at a time by enlarging the gel area and forming many grooves (wells) in the upper part of the gel. However, there is a problem that the separation speed is low and the analysis takes time. Further, in order to analyze the gel after the separation of the target substance, it is necessary to remove the gel from the electrode tank, stain and analyze the gel, and there is a problem that it takes time and effort due to a complicated operation.

On the other hand, in the capillary electrophoresis method proposed for high-speed analysis, it is possible to relatively easily detect a separated substance, but it is possible to separate only one sample per capillary, There was a problem that the reproducibility for each used capillary was poor. Therefore, the fact is that plate gels are still used for analysis of multiple samples such as DNA sequences.

Therefore, in the separation and analysis of DNA, protein, etc., many samples can be analyzed at once with high speed and high accuracy.
A method capable of separating and analyzing has been desired. Accordingly, the invention of this application has been made in view of the circumstances described above, and it is an object of the present invention to solve the problems of the prior art and to provide a micro electrophoresis device having high separation efficiency and high detection sensitivity. Is an issue.

[0011]

Means for Solving the Problems According to the invention of the present application, to solve the above-mentioned problems, first, a concentrated gel layer having a thickness of at least 50 μm to 350 μm and a thickness of 50 μm are provided.
Provided is an ultra-small thin-layer gel electrophoresis apparatus characterized in that a flat gel comprising a separation gel layer of μm or less is used as a support.

Secondly, the invention of the present application is directed to the ultra-small thin-layer gel electrophoresis apparatus, wherein the separation gel layer of the flat polymer gel has a height higher than the bottom of a pair of opposing translucent plates. The polymer gel solution is injected into the gap between the light-transmitting plates by capillary action to be formed by immersion in the molecular gel solution, and thirdly, the polymer gel solution is transferred to the light-transmitting plate. The gap between the light-transmitting plates is constant below the height position and rapidly widens above the height position so as to be injected by capillary action to the desired height position between them. This is provided as an embodiment.

Fourth, the invention of the present application also provides that the detector in the ultra-small thin-layer gel electrophoresis apparatus is a thermal lens microspectroscope.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The ultra-small thin-layer gel electrophoresis apparatus of the invention of the present application is an extremely thin and compact thin-layer gel electrophoresis apparatus comprising a concentrated gel layer having a thickness of 50 to 350 μm and a separation gel layer having a thickness of 50 μm or less. The gel is used as a support. By reducing the size of the gel, the protein or D
It is possible to separate the NA at high speed, which simplifies the time-consuming and time-consuming detection operation.
An ultra-small thin-layer gel electrophoresis apparatus to which a high-resolution method can be applied can be obtained.

In order to make the gel serving as a support smaller, it is necessary to make the sample band as narrow as possible. Therefore, a concentrated gel layer for concentrating the sample to form a thin layer is provided, and the concentrated target substance is separated in the separation gel layer.

The concentrated gel discontinuously changes ions having high mobility (lead ions) in the sample-loaded gel and ions (final ions) having lower mobility than the sample molecules in the electrode buffer solution above the gel. By causing the highest voltage gradient to be applied to the sample molecule between these two ions, the loaded protein is concentrated in an extremely thin layer. The concentrated sample molecules are separated in a separation gel having a concentration suitable for the molecular weight of the component to be separated by a difference in the mobility of each component.

If the gel is too thick, it is difficult to separate it with a small amount of sample, and it is necessary to increase the area of the gel. Therefore, the gel is preferably as thin as possible. However, if it is too thin, on the contrary, it becomes impossible to inject a certain amount of the sample or the gel becomes difficult to handle, which is not preferable. Therefore, in the ultra-small thin-layer gel electrophoresis apparatus of the invention of this application, the thickness of the gel is preferably 50 to 350 μm for the concentrated gel layer and 50 μm or less for the separation gel layer. The concentrated gel layer has a thickness of 50 to 350 μm, more preferably 200 to 350 μm, in order to make it difficult to form a well for injecting a sample or to inject a sample solution.
μm. With this thickness, it is possible to inject a fixed amount of the sample solution into a well on the gel formed between a pair of translucent plates using a micropipette used in ordinary chemical experiments. In addition, the thickness of the concentrated gel layer is thicker than the separation gel layer, and even if it is constant, the portion where the sample is injected is thicker, and becomes thinner as it approaches the separation gel layer,
At the interface with the separation gel layer, both may have the same thickness. In the ultra-small thin-layer gel electrophoresis apparatus of the invention of this application, it is preferable that the sample inlet portion is thick and has a shape that becomes thinner as approaching the separation gel layer. This relates to a preferred method for producing a flat gel in the ultra-small thin-layer gel electrophoresis apparatus of the present invention described later.

On the other hand, it is preferable that the separation gel layer is as thin as possible. If the gel is too thick, the separation speed becomes slow, a large amount of sample is required, and the method of detecting separated sample molecules is limited, which is not preferable. If it is 50 μm or less, it becomes possible to incorporate not only a conventional detector and a detection method but also a new detection method such as a thermal lens.
It is suitable.

In the invention of this application, the total area of the concentrated gel layer and the separated gel layer is not particularly limited. However, in order to facilitate the handling of a thin gel and the separation with a small amount of material, the size is preferably 5 cm × 5 cm or less. The smaller the size of the condensed gel, the faster the passing speed of the sample molecules can be.However, if the size is too small, the concentration may not be sufficiently performed before reaching the separation gel, and the detection sensitivity may be reduced. . Therefore, the width is 5 cm or less, more preferably 1 to 5 cm, and the height is 0.5 to 1 c.
m is preferable. On the other hand, the size of the separation gel is
The width is preferably 5 cm or less, more preferably 1 to 5 cm, which is the same as that of the concentrated gel, and the height is 3 cm or less, more preferably 1 to 2.5 cm. Of course, the sizes of the concentrated gel layer and the separation gel layer in the ultra-compact thin-layer gel electrophoresis apparatus of the invention of this application are not limited to these, and are within a range that does not impair ease of handling and separation ability. Can be set with.

As the gel polymer, a polyacrylamide gel having a high separation ability is generally used. The characteristics of polyacrylamide gel are (1) transparent and easy to detect sample molecules (protein or DNA) after staining, and (2) electro-osmosis because of no charge in the molecule. (The phenomenon that the solution as a whole moves to the electrode side with the opposite sign due to the charge) does not occur. Usually, acrylamide is used as a reagent for forming a linear polymer, and N, N'-methylenebisacrylamide (hereinafter, referred to as "N, N'-methylenebisacrylamide") is used.
(referred to as bis) as a reagent for cross-linking between linear chains, and polymerized from a mixed aqueous solution. Acrylamide and bi in solution for polymerization
s weight% (T%) and bis weight% (C
%) Is generally called the gel density and the degree of cross-linking, respectively. When two or more layers of a gel for concentration and a gel for separation are used, usually, C is made equal and gels differing only in T are used.

In the ultra-small thin-layer gel electrophoresis apparatus of the present invention, the flat gel of the support is formed between a pair of opposing light-transmitting plates. FIG. 1 exemplifies a plate gel as a support of the ultra-small thin-layer gel electrophoresis apparatus of the invention of this application, and specifically describes a method for producing the same. In the invention of this application, a separator (3) is inserted between a pair of opposing translucent plates (1) and (2), and a gel solution is injected between the translucent plates to obtain a gel (4). .

As the light-transmitting plate, different ones can be selected according to the method of detecting sample molecules used in the ultra-compact thin-layer gel electrophoresis apparatus of the present invention. Any material may be used as long as it can transmit such light without absorbing or reflecting light such as ultraviolet light, visible light, infrared light, and laser light used in the detection method. . In addition, any material that is stable to the above light, has a uniform surface, and has an appropriate strength that can serve as a cell when forming a gel, such as a plastic plate of acrylic or the like, a glass plate, a quartz plate, etc. Is exemplified. Preferably, the glass plate is high in strength, transparent, stable to light, and inexpensive.

The gel solution is prepared by diluting a gel polymerized by an ordinary polymerization method, adding a polymerization initiator or the like to a monomer solution, and polymerizing between the light-transmitting plates (1) and (2). Or cause a reaction. For example, acrylamide (4 to 20 g in 100 ml of solution), crosslinking agent N, N '
-Methylene bisacrylamide (about 5% of acrylamide by weight), an aqueous solution mixed with an amphoteric electrolyte mixture,
Deaeration, cooling, polymerization initiator (ammonium persulfate), polymerization accelerator (N, N, N ', N'-tetramethylethylenediamine)
Is generally added between the transparent plates (1) and (2) and solidified. Of course, the solution concentration, the crosslinking agent, the initiator, the polymerization accelerator and the like are not limited to these. The concentrated gel layer (5) and the separated gel layer (6)
For example, after the separation gel layer (6) is gelled by the above method or the like, the raw material solution of the concentrated gel layer (5) is injected, and the concentrated gel layer (5) is formed on the separated gel layer (6). Two layers. Of course, the order and method of gelation are not limited to this.

In the invention of this application, since the thickness of the gel to be produced is particularly thin, that is, 50 μm or less, it is necessary to prepare a raw material solution of the gel and then inject the gel solution using a syringe or a pipette by an ordinary method. Is difficult. Therefore, the bottoms of a pair of opposing translucent plates (1) and (2) whose intervals are fixed by the separator (3) are immersed in a gel solution, and the translucent plate (1) is formed by capillary action. ,
It is preferable to inject during (2).

At this time, the interval between the light-transmitting plates (1) and (2) is equal to the thickness of the intended separation gel layer (6), and the height position of the desired separation gel layer (6) from the bottom portion Until is constant. Then, above the height position, the interval between the light-transmitting plates (1) and (2) increases rapidly. This allows
The gel solution that has risen due to the capillary action stops at the height of the desired separation gel layer (6) and solidifies,
The interface with the concentrated gel layer (5) formed on the separation gel layer (6) becomes horizontal and uniform. As described above, as a method of rapidly increasing the interval between the light-transmitting plates (1) and (2) from a certain position, both the light-transmitting plates (1) and (2) spaced at a fixed interval are used. Alternatively, a method of sharply inclining one side from the height position to widen the gap, or digging a groove in one or both of the translucent plates (1) and (2) at the height position, as a result There is a method of widening the interval between the two. Preferably, one of the light-transmitting plates (2) is processed diagonally from above to the light-transmitting plate (1) obliquely downward from above to the target height of the separation gel layer (6). This is a method of providing an inclined portion (7). By this method,
The inclined portion (7) of the translucent plate (2), that is, the vertical section (8) is an inverted triangle, and the vertical section (8) of the inverted triangle is an inverted triangle having the same area in any part. Will be shown. That is, the concentrated gel layer (5) formed on the inclined portion (7) has a constant vertical cross-sectional area in the thickness direction. As a result, the sample injected into any position
It is concentrated under the same conditions and can reach the separation gel layer (6). When the distance between the light-transmitting plates is increased by this method, the height of the light-transmitting plates (1) and (2) may be the same. (1) is preferably higher than (2) so as to make the injection easier. Further, the processing for widening the interval between the inclined portion (7) and the translucent plate exemplified above can be performed by using a normal glass processing method such as cutting, polishing, or cutting.

The distance between the light-transmitting plates (1) and (2) is the largest at the opening (9), and the distance between the light-transmitting plates (1) and (2) at the opening is: After the separation gel layer (6) is solidified, the thickness is preferably 350 μm or less, more preferably 200 to 500 μm, in order to inject a gel solution from the opening (9) to form a concentrated gel layer (5). It is 300 μm. This makes it easy to inject the gel solution with a micropipette or the like.

The distance between the transparent plates (1) and (2) is
Determined by the separator (3). Regarding the size of the separator, it is sufficient that the thickness is equal to the thickness of the desired separation gel layer (6), and the height is preferably at least the same as or larger than the translucent plate (2). Separator (3) and translucent plate (1), (2)
Must be sealed and function as a cell for making gel so that the gel solution to be injected does not leak. For example, a separator (3) and a light-transmitting plate (1), (2)
May be fixed with an adhesive or the like, may be fitted through a packing or the like, and may be sealed. Of course, the separator (3) and the translucent plates (1) and (2) may be integrated into a cell whose thickness is limited in advance. However, in order to easily remove the gel from the translucent plates (1) and (2) later, it is preferable that they are individually provided.

Further, in the upper part (sample injection part) of the concentrated gel layer near the opening part (9), a comb-shaped plate (comb) according to the width and thickness at the time of gel preparation is put and solidified. ) Can be formed. This well (10)
By increasing the number, it is possible to separate and analyze a large number of specimens at one time. However, if the number is too large, the distance between the wells (10) is shortened and the separation ability is unfavorably affected. It may be prepared according to the width of the gel (4) to be used. At the time of gel electrophoresis measurement, a sample is injected into a well (10) prepared using a micropipette, so that a sample of several nL can be separated and analyzed. The relationship between the amount of the sample and the sensitivity of the detection method is important, and it is necessary to adjust the amount of the target molecule in the sample volume to an appropriate value.

The gel (4) from which the sample has been separated is stained or used for the purpose of fixing sample molecules (protein or DNA), removing the amphoteric electrolyte mixture from the gel, and further facilitating detection. Preferably, a label is introduced. Examples of the dyeing method include dye dyeing and dyeing with a metal such as silver. In the ultra-small thin-layer gel electrophoresis of the invention of this application, various staining methods can be used for the gel (4), and those suitable for the detection method can be selected. Of course, some detection methods do not require staining or labeling, and when such a detection method is applied, staining may not be performed.

In the ultra-small thin-layer gel electrophoresis apparatus of the invention of the present application, the detector comprises a trace amount of protein and DNA.
Any method can be used as long as it can be detected with high accuracy, and commonly used methods such as radioisotope reading, ultraviolet absorbance analysis, and fluorescence analysis can be used. Preferably, it is a thermal lens microscope having a high spatial resolution of 2 μm and capable of detecting a non-luminescent substance with ultra-high sensitivity.

The thermal lens phenomenon is a phenomenon observed when a cell containing a solution is irradiated with a laser beam. In a solution, when absorbing the wavelength of laser light, energy is stored in a form along the intensity distribution of the laser light. Without the fluorescence process, this energy is transferred to the solvent as heat. Normally, since the change in the refractive index of the solvent with respect to the temperature is negative, the effect appears as if a concave lens had appeared in the cell. In general, since the laser is slightly dispersed, the beam radius gradually increases, resulting in an extreme decrease in sensitivity.Therefore, a stable lens is provided by placing a convex lens for focusing the laser light in front of the sample cell. A thermal lens can be obtained.

In the ultra-small thin-layer gel electrophoresis of the invention of this application, sample molecules can be detected with high accuracy by using a thermal lens microscope as a detector. The thermal lens microscope uses a single-beam system that uses a single laser that combines both the dual-beam system, in which an induction laser for generating a thermal lens and a probe laser for monitoring the thermal lens are separate. There may be. Preferably,
It is preferable to use a dual beam system in which a laser for inducing a thermal lens is used as a dye laser to measure an absorption spectrum with respect to a wavelength or use a pulse laser. As the probe light, a low-output He-Ne laser or the like can be used, whereby the noise level can be reduced to 1/10 or less.

When used as a detector of the ultra-small thin-layer gel electrophoresis apparatus of the present invention, as shown in FIG. 2, an excitation laser beam (11) and a probe (1) are used.
The probe light (13) from 2) is condensed by the objective lens (14), the sample band (15) of the stained gel (4) is scanned, and the probe scattered by the thermal lens (16) emitted by the excitation light. The light (13) is passed through a pinhole (17), the excitation light is removed by an excitation light cut filter (18), and only the probe light (13) is collected by a condenser lens (19). 20).

As a detector of the ultra-small thin-layer gel electrophoresis apparatus of the invention of this application, a method of detecting a DNA fragment separated in a monochromatic or multicolor system using a DNA replication reaction of an enzyme is also used. It has been known. That is, a DNA fragment whose sequence is to be determined is incorporated into a vector, propagated, the vector-DNA is separated and purified, and a fluorescently labeled short DNA is used.
A fragment (primer) is bound, and based on the fragment, a DNA chain having a complementary sequence is extended using an enzyme. The lower part of the migration path of the separation band obtained by preparing {A}, {C}, {G}, and {T} based on the difference in the ends, filling these into separate wells, and performing electrophoresis, is an image multiplying machine. In addition, a DNA fragment spectrum is obtained from the position of the electrophoresis path which receives and emits light with a video camera and the temporal change of the fluorescence peak intensity, and the sequence is automatically determined by a computer.

Of course, the detection method in the ultra-small thin-layer gel electrophoresis apparatus of the invention of the present application is not limited to those exemplified above, and may be a detection method used by conventional gel electrophoresis such as spectroscopy. Good.

The embodiments of the present invention will be described in more detail with reference to the accompanying drawings. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail.

[0037]

EXAMPLES Example 1 (1) Preparation of Discontinuous Polyacrylamide Gel FIG. 1 illustrates a flat gel as a support of the ultra-compact thin-layer gel electrophoresis apparatus of the present invention.

Acrylamide is used as a reagent for forming a linear polymer, and N, N'-methylenebisacrylamide (hereinafter referred to as bis) is used as a reagent for cross-linking between linear chains.
As 2), an amide gel was prepared using a slide glass. (Concentrated gel layer (5): length 5 mm, 3% T, 3
% C, buffer pH 6.8 Tris / HCl, thickness: 50 to 350
μm; separation gel layer (6): length 70 mm, 7.5% T,
(3% C, buffer pH 8.8 Tris / HCl, thickness: 50 μm) A comb (10) was prepared by adding a comb. (2) Gel electrophoresis of DNA A 100 base pair ladder (bpl) DNA sample was used. Electrophoretic separation was carried out in an electrode tank buffer using a pH 8.3 Tris / Glycine solution in an electric field of 30 to 120 V / cm. (3) Detection of Separated DNA by Thermal Lens The DNA separation pattern was stained with silver and detected with a thermal lens microscope (FIG. 2). An air-cooled Ar + laser with a wavelength of 488 nm and an output of 4 mW was used for the excitation light (11) of the thermal lens microscope, and the probe light (13) was at a wavelength of 633 nm and an output of 1 m.
A W He-Ne laser was used. Comparative Example 1 (1) Preparation of discontinuous polyacrylamide gel A conventional method using acrylamide as a reagent for forming a linear polymer and N, N'-methylenebisacrylamide (hereinafter referred to as bis) as a reagent for crosslinking between linear chains. Thus, a polyacrylamide gel was formed between glass plates. (Separation gel layer: length 1
50 mm, 7.5% T, 3% C, buffer pH 8.8 Tris
/ HCl, thickness 350 μm) A comb (10) was prepared by inserting a comb. (2) Gel electrophoresis of DNA As in Example 1, 100 base pair ladder (bpl) DN
Sample A was used. PH 8.3 Tris /
Using a Glycine solution, electrophoretic separation was performed in an electric field of 30 to 120 V / cm. (3) Detection of Separated DNA by Thermal Lens In the same manner as in Example 1, the DNA was separated by a thermal lens microscope.
An NA sample was detected.

As shown in FIG. 3, the bandwidth of the DNA sample obtained by the ultra-small thin-layer gel electrophoresis apparatus of the invention of this application is 100 μm, which is about 1/10 that of normal gel electrophoresis. It was width. The migration distance required for the separation was 18 mm, and the time required for the separation was 15 minutes.
On the other hand, normal gel electrophoresis takes 2.5 hours,
A migration distance of 30 mm was required. Each of the separated sample bands is visible to the naked eye only up to about 1500 bp, but is measured at 2400 bp by using a thermal lens microscope.
Could be detected up to

Further, in the ultra-compact thin-layer gel electrophoresis apparatus of the present invention, the gel separation ability (the number of theoretical plates: 2)
6,000 to 116,000) indicates the gel separation ability (theoretical plate number: 48,000 to 8) in conventional gel electrophoresis.
5,000).

By using a thermal lens microscope, it is possible to clearly decompose a portion of 1000 base pairs or more, and by this detection method, it was possible to accurately detect a narrow band.

[0042]

As described above in detail, according to the present invention, a new ultra-small thin-layer gel electrophoresis apparatus capable of separating a target molecule at high speed, with high accuracy, and easily can be provided.

[Brief description of the drawings]

FIG. 1 is a diagram exemplifying a flat gel of a microminiature thin-layer gel electrophoresis apparatus of the present invention.

FIG. 2 is a diagram exemplifying a thermal lens microscope as a detector of the microminiature thin-layer gel electrophoresis apparatus of the present invention.

FIG. 3 is a diagram showing a result of gel electrophoresis analysis of a DNA sample in an example of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Translucent board 2 Translucent board 3 Separator 4 Gel 5 Concentrated gel layer 6 Separation gel layer 7 Inclined part 8 Longitudinal section 9 Opening 10 Well 11 Excitation laser beam 12 Probe 13 Probe beam 14 Objective lens 15 Sample Band 16 Thermal lens 17 Pinhole 18 Excitation light cut filter 19 Condenser lens 20 Photodiode

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12N 15/09 G01N 27/26 315E 4B063 C12Q 1/68 315B 325A C12N 15/00 A (72) Inventor Shiro Sawada Tokyo 6-37-2-504 Minami-Senju, Arakawa-ku (72) Inventor Jung Kim Key 4-4-1-611 Hikonari, Misato-shi, Saitama F-term (reference) 2G040 AA02 AB07 BA24 BB07 CA11 CA13 CA23 EA06 2G043 AA06 BA16 CA03 DA01 EA01 EA13 EA19 HA01 JA03 KA02 KA03 KA05 KA09 LA01 LA03 LA05 2G059 AA10 BB12 CC16 DD01 EE01 FF12 GG01 GG03 GG08 HH02 HH03 HH06 JJ02 JJ11 QKKFA KK04 QQ79 QR82 QS16 QS39 QX02

Claims (4)

[Claims] 1. An ultra-small thin-layer gel electrophoresis apparatus characterized in that a flat gel composed of a concentrated gel layer having a thickness of at least 50 μm to 350 μm and a separation gel layer having a thickness of 50 μm or less is used as a support. 2. A separation gel layer of a flat polymer gel is formed by immersing the bottom portions of a pair of opposing translucent plates in a polymer gel solution, whereby the polymer gel solution is translucent by capillary action. 2. The ultra-small thin-layer gel electrophoresis device according to claim 1, wherein the electrophoresis device is prepared by injecting a liquid between the plates. 3. The interval between the light-transmitting plates is below the height so that the polymer gel solution is injected by capillary action to a desired height between the light-transmitting plates. 3. The ultra-small thin-layer gel electrophoresis device according to claim 1, wherein the device is constant and rapidly widens at the height position or higher. 4. The microminiature thin-layer gel electrophoresis apparatus according to claim 1, wherein the detector is a thermal lens microspectroscope.