Electrophoresis cartridge and electrophoresis method

Abstract

The present invention can form holes without comb teeth, can use a gel having high convenience and degree of freedom, and can prevent degradation of sample separation performance due to swelling of the gel. An electrophoresis cartridge (1) is provided with: a gel tank (2); a gel (7) accommodated in the gel tank (2); a buffer tank (3) that is continuous with the gel tank (2) and into which a buffer is injected, the buffer tank (3) being a tank into which a sample (target biological substance) to be subjected to electrophoresis in the gel (7) is injected; and oil (8) in contact with the gel (7) of the gel tank (2). And the gel (7) and the oil (8) are pre-encapsulated in the electrophoresis box (1).

Description

Technical Field

[0001] The present disclosure relates to an electrophoresis cassette and an electrophoresis method, and relates to an electrophoresis cassette pre-packaged with a gel and an electrophoresis method using the electrophoresis cassette. Background Art

[0002] A gel electrophoresis method is known, which utilizes the phenomenon that when an electric field is applied to a substance with a charge, the substance moves in the direction of an electrode of opposite polarity to analyze biological substances such as nucleic acids and proteins. Usually, as a support for biological substances, electrophoresis gels such as agarose gel and acrylamide gel are used. The moving speed in the electrophoresis gel is different according to the molecular weight of the biological substance, so the biological substance is separated into different bands according to the molecular weight. The gel electrophoresis method has high resolution for the separation of biological substances, and is therefore also used to measure the fragment length of DNA to grasp the state of the specimen. In addition, by fluorescently labeling DNA and observing its brightness, it is also used for the quantification of DNA of a specific length.

[0003] Generally, the electrophoresis gel is prepared and used before each electrophoresis. However, in the preparation of the electrophoresis gel, if it is an agarose gel, the gel needs to be dissolved, and if it is a polyacrylamide gel, the reagent needs to be adjusted and degassed, which is a problem of a lot of man-hours.

[0004] As a pre-prepared prepackaged polyacrylamide gel that can be immediately subjected to electrophoresis without adjusting the electrophoresis gel, Patent Documents 1 and 2 disclose a method called slab gel, in which the gel is formed between glass plates and stored in a low pH buffer.

[0005] In addition, Patent Document 3 discloses an electrophoresis apparatus in which a fluorescent dye is added to a pre-prepared pre-packaged gel, thereby eliminating the need for fluorescent staining of a sample.

[0006] In addition, Patent Document 4 discloses an electrophoresis cassette in which a gel and a buffer solution are prepackaged.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: U.S. Patent No. 7422670

[0010] Patent Document 2: U.S. Patent No. 6783651

[0011] Patent Document 3: U.S. Patent No. 8562802

[0012] Patent Document 4: U.S. Patent No. 8591713 Summary of the invention

[0013] Problems to be solved by the invention

[0014] However, the pre-packaged gel requires a structure called a pore for receiving a sample, and there is a problem that there are restrictions on maintaining this structure.

[0015] In many prepackaged gels, a resin plate called a comb is inserted into the gel, and the comb is pulled out before use, and the sample is injected into the hole formed thereon for use (Patent Documents 1, 2, and 3). In an electrophoresis automation device, when a box with these prepackaged gels is used, a comb removal mechanism is required. In addition, when an operator manually removes the comb before using the electrophoresis automation device, since the hole will be exposed in the device for a certain period of time before the start of electrophoresis, contamination and drying of the buffer portion may occur.

[0016] Patent document 4 discloses an electrophoresis box in which a buffer in contact with a gel is pre-packaged and the holes are held without comb teeth. However, in order to prevent swelling of the gel during storage, the gel composition is limited to a cross-linked gel. In addition, among cross-linked gels, a composition with less swelling needs to be selected. In addition, in order to prevent swelling and shrinkage caused by osmotic pressure difference, the salt concentration of the buffer and the gel needs to be consistent.

[0017] Therefore, an object of the present disclosure is to provide an electrophoresis cassette and an electrophoresis method, which can form holes without comb teeth, can use a gel with high convenience and freedom, and can prevent a decrease in separation performance of a sample due to swelling of the gel.

[0018] Methods for solving problems

[0019] In order to solve the above problems, the electrophoresis cassette of the present disclosure includes a gel tank for storing a gel and a buffer tank for injecting a buffer solution. The buffer tank is a tank continuous with the gel tank. The buffer solution is injected into a sample to be electrophoresed in the gel. The electrophoresis cassette is prepackaged with the gel and oil in contact with the gel.

[0020] In order to solve the above problems, the electrophoresis method disclosed herein includes:

[0021] preparing an electrophoresis box, the electrophoresis box having a gel tank for storing a gel and a buffer tank for injecting a buffer, and pre-packaging a gel and oil in contact with the gel, the buffer tank being a tank continuous with the gel tank, the buffer being injected into a sample to be electrophoresed in the gel;

[0022] Add buffer into the buffer tank of the electrophoresis box to separate the oil from the gel;

[0023] injecting the sample into the buffer solution put into the buffer solution reservoir; and

[0024] An electric field is applied to the sample, causing electrophoresis of the sample in the gel.

[0025] Effects of the Invention

[0026] According to the electrophoresis cassette and the electrophoresis method of the present disclosure, holes can be formed without comb teeth, a gel with high convenience and freedom can be used, and a reduction in separation performance of a sample due to swelling of the gel can be prevented. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a front view of the electrophoresis cassette according to the embodiment.

[0028] Figure 2 It is a cross-sectional view of the electrophoresis cassette according to the embodiment.

[0029] Figure 3 This is a graph illustrating the results of a swelling test of a gel in a case where a buffer solution is layered in a comparative example.

[0030] Figure 4 This is a graph showing the results of a swelling test of the gel in Example 1 when the oil was separated into layers.

[0031] Figure 5 This is a diagram showing the electrophoresis image of Example 2. DETAILED DESCRIPTION

[0032] In all the drawings used to illustrate the present embodiment, the same reference numerals are used for parts having the same function, and their repeated descriptions are sometimes omitted. In addition, the present invention is not limited to the description of the embodiments shown below. The present invention is defined by the attached claims, but it is easy for those skilled in the art to understand that the specific configuration can be changed without departing from the scope of its ideas or purpose.

[0033] In order to facilitate understanding of the invention, the position, size, shape, range, etc. of each component shown in the drawings and the like may not represent the actual position, size, shape, range, etc. Therefore, the present invention is not necessarily limited to the position, size, shape, range, etc. disclosed in the drawings and the like.

[0034] In this specification, unless otherwise specified, a constituent element expressed in the singular includes the plural form.

[0035] use Figure 1 and Figure 2 The electrophoresis cell 1 according to the embodiment of the present disclosure will be described. Figure 1 and Figure 2 1 is a diagram showing the structure of the electrophoresis box 1 according to the embodiment. Figure 2In the example of FIG. 1 , a support 9 covering the gel 7 is provided. The support 9 is resistive (including an insulating support and a support with a large resistance (including not only a complete insulator but also a support that is slightly energized)).

[0036] The electrophoresis cell 1 includes an upper electrode 4a and a lower electrode 4b for applying a voltage. A power source (not shown) is connected to the upper electrode 4a and the lower electrode 4b, so that a voltage can be applied between the pair of electrodes 4a and 4b. Although not shown, a voltage control device for controlling the operation of the power source may also be connected to the power source.

[0037] In addition, the following description will be given by taking as an example the case where the sample to be recovered (target biological substance) is nucleic acid.

[0038] Gel 7 is a separation medium for separating target biological substances from unnecessary substances. As gel 7, for example, a known gel such as agarose gel or polyacrylamide gel can be used. The thickness of gel 7 is not particularly limited, but is preferably 0.5 to 18 mm from the viewpoint that the bands of biological substances obtained by electrophoresis are clear and easy to visually recognize. In addition, the thickness of gel 7 may not be constant.

[0039] The electrophoresis box 1 includes a gel tank 2 capable of storing a gel 7. The gel tank 2 is provided with an injection port 5 for injecting the gel 7. The gel 7 is pre-packed in the gel tank 2.

[0040] The electrophoresis box 1 includes a buffer tank 3 that can store a buffer solution, and the buffer solution is injected into a sample (target biological substance) to be electrophoresed in the gel 7. The buffer tank 3 is a tank continuous with the gel tank 2. The internal space of the buffer tank 3 is spatially continuously connected to the internal space of the gel tank 2. Before electrophoresis, a buffer solution is injected into the buffer tank 3. The buffer solution is not pre-packaged.

[0041] The gel 7 contained in the gel tank 2 is in contact with the oil 8. The oil 8 is pre-packaged in the buffer tank 3. The oil 8 is a liquid that is phase-separated from the water and the gel 7, and can be, for example, a non-polar solvent, a low-polarity solvent, or a lipid. The oil 8 includes benzene and toluene. The interfacial tension between the oil 8 and the water and the gel 7 is such that the phase separation state can be maintained, and can be, for example, set to 10 mN / m or more. In addition. The surface tension of the oil 8 can be set to, for example, 10 mN / m. In addition, the interfacial tension between the water, the gel 7, and the oil 8 can be smaller than the surface tension of the oil 8.

[0042] The specific gravity of the oil 8 is smaller than that of the buffer solution, so that the interface between the buffer solution and the gel 7 can be easily formed during electrophoresis.

[0043] The support body 9 is formed of resin or the like. By providing an opening in the buffer tank 3 or providing a perforating mechanism, a sample containing a target biological substance to be separated can be injected into the buffer tank 3. In this embodiment, an opening for injecting a sample toward the upper surface is formed at one end of the buffer tank 3 or in the vicinity thereof.

[0044] The target biological substance is injected into the buffer tank 3 in the form of an injection solution mixed with a liquid having a specific gravity greater than that of the buffer solution. Examples of solvents for mixing the target biological substance include glycerol aqueous solution and sucrose water. When the solvent is glycerol aqueous solution, the glycerol concentration can be, for example, 6%. The viscosity of the injection solution can be, for example, 1 mPa·s.

[0045] The distance between the buffer reservoir 3 and the lower electrode 4 can be set arbitrarily. The distance can be appropriately designed according to the composition of the gel 7 (eg, gel concentration), the molecular weight of the target biological substance, and the like.

[0046] In this embodiment, the gel tank 2 and the buffer tank 3 are roughly rectangular parallelepiped, but their structure, shape, size, etc. are not limited to those shown in the figure. The structure, shape, size, etc. of the gel tank 2 and the buffer tank 3 can be set arbitrarily. The width dimension of the gel tank 2 and the buffer tank 3 (i.e., the dimension in the horizontal direction orthogonal to the direction of the electric field. Not shown) Figure 2 ) can be equal or different.

[0047] In the present embodiment, the migration direction of the biological substance is the vertical direction (Z direction in the figure), and the gel tank 2 and the buffer tank 3 are arranged continuously in the vertical direction. However, the migration direction is not limited to the vertical direction.

[0048] (Electrophoresis method)

[0049] Next, an electrophoresis method using the electrophoresis cassette 1 will be described.

[0050] First, prepare an electrophoresis box 1. The electrophoresis box 1 includes a gel tank 2, a gel 7 stored in the gel tank 2, a buffer tank 3 into which a buffer is placed, and an oil 8 in contact with the gel 7 in the gel tank 2, wherein the buffer tank 3 is a tank continuous with the gel tank 2, and the buffer is poured into a sample to be electrophoresed in the gel 7. The gel 7 and the oil 8 are pre-packaged in the electrophoresis box 1.

[0051] Next, a buffer solution is added to the buffer solution tank 3 of the electrophoresis box 1 to separate the oil 8 from the gel 7 .

[0052] Then, the sample is injected into the buffer solution placed in the buffer solution tank 3 .

[0053] Finally, an electric field is applied to the sample to cause electrophoresis of the sample in the gel 7 .

[0054] (Comparative Example)

[0055] Hereinafter, comparative examples will be described.

[0056] Acrylamide and cross-linking agent were prepared to a total monomer content of 12% (w / v), and polymerized in a 2 mL tube to form a gel. Tris Glycine was used as the buffer. Figure 3 Methylenebisacrylamide, dihydroethylenebisacrylamide, and bisacryloylcystamine were used in the weight ratio described in. Equal amounts of buffer Tris Glycine of the same concentration were injected onto the above gel.

[0057] The buffer was removed by decantation every few days and the weight of the gel was measured. After measurement, the decanted buffer was returned to the original tube and stored again. This process was repeated until 38 days later.

[0058] The results are shown in Figure 3 The graph is plotted by weight swelling ratio with respect to time (with the initial swelling ratio as 100%). It is found that the swelling amount increases when the ratio of the crosslinking agent is small.

[0059] (Example 1)

[0060] Next, Example 1 will be described.

[0061] After preparing the gel layer in the same manner as in the comparative example, oil or buffer was injected into the tube to evaluate the weight swelling rate. The gel was prepared by making acrylamide and a crosslinker so that the total monomer amount was 12% (w / v), and dihydroethylenebisacrylamide was used as the crosslinker at a weight ratio of 149:1 to acrylamide. Tris Glycine prepared to pH 9.5 or Bis-Tris Tricine BES prepared to pH 6.9 was used as the buffer. Mineral oil, silicone oil or toluene was used as the oil.

[0062] The results are shown in Figure 4 When the buffer solution was injected into the tube, the tube swelled by 15% to 35% within 6 days, but when the oil was injected into the tube, the swelling rate was less than 5% within 6 days.

[0063] (Example 2)

[0064] Next, Example 2 will be described.

[0065] Acrylamide and cross-linking agent were prepared to a total monomer amount of 12% (w / v), and the gel was polymerized in a support body with a flow path structure formed on a PET sheet to form a gel. Dihydroethylenebisacrylamide was used as a cross-linking agent at 6% (w / w) of acrylamide. Tris Glycine was used as a buffer. Buffer or oil was injected into the buffer tank on the gel, stored at 37°C for 4 days, and then electrophoresed. A 100bp ladder was used for the electrophoresis sample.

[0066] Electrophoresis images such as Figure 5 As shown. Compared with the situation just after the injection of the buffer solution (0 day, buffer solution) 4 days after the injection of the buffer solution (4 days, buffer solution), the pores of the gel became larger and the migration became faster. In contrast, the situation 4 days after the injection of the oil (4 days, mineral oil and silicone oil) showed the same migration state as the situation just after the injection of the buffer solution (0 day, buffer solution).

[0067] (Effects of Embodiments)

[0068] In this embodiment, the gel 7 is pre-packaged in the gel tank 2, and the oil 8 in contact with the gel 7 in the gel tank 2 is pre-packaged, so that the holes can be formed without comb teeth. Therefore, it is not necessary to install a comb tooth removal mechanism. In addition, since the gel 7 and the oil 8 are pre-packaged, it is possible to prevent contamination and drying of the buffer portion.

[0069] In addition, in the present embodiment, since the composition of the gel 7 for preventing the swelling of the gel 7 is not limited, the gel 7 with high convenience and freedom can be used.

[0070] Furthermore, in the present embodiment, by providing the oil 8 in contact with the gel 7 , it is possible to prevent a decrease in the separation performance of the sample due to swelling of the gel 7 .

[0071] In addition, in the present embodiment, it is necessary to inject a buffer solution before electrophoresis, but this is the same dispensing operation as the injection liquid (mixed solution of a sample and a solvent), and can therefore be easily performed.

[0072] In this embodiment, since the specific gravity of the oil 8 is smaller than that of the buffer solution, the oil 8 can be displaced to the upper side by injecting the buffer solution into the buffer solution reservoir 3 when the electrophoresis cell 1 is used, thereby forming a gel 7-buffer interface and performing electrophoresis.

[0073] In addition, in this embodiment, since the buffer is injected before electrophoresis, the salt concentrations of the gel 7 and the buffer can be greatly changed, and the difference in conductivity between the buffer and the gel 7 can be used to concentrate the band and improve the separation performance.

[0074] It should be noted that the present disclosure is not limited to the above-mentioned embodiments, and includes various modified examples. For example, the above-mentioned embodiments are described in detail in order to easily explain the present disclosure, and are not necessarily limited to having all the described structures. In addition, with respect to a part of the structure of each embodiment, other structures can be added, deleted, or replaced.

[0075] Description of Reference Numerals

[0076] 1 ... electrophoresis box, 2 ... gel tank, 3 ... buffer tank, 4a ... electrode, 4b ... electrode, 5 ... injection port, 7 ... gel, 8 ... oil, 9 ... support body.

Claims

1. An electrophoresis box, characterized in that: have: a gel tank for storing gel; and A buffer tank is filled with a buffer solution, the buffer tank is a tank continuous with the gel tank, and the buffer solution is injected into the sample to be electrophoresed in the gel, The electrophoresis box is pre-packaged with the gel and oil in contact with the gel.

2. The electrophoresis box according to claim 1, characterized in that: The interfacial tension between the gel and the oil is 10 mN / m or more.

3. The electrophoresis box according to claim 1, characterized in that: The surface tension of the oil is 10 mN / m or more.

4. The electrophoresis box according to claim 1, characterized in that: The interfacial tension between the gel and the oil is lower than the surface tension of the oil.

5. The electrophoresis box according to claim 1, characterized in that: The specific gravity of the oil is lower than that of the buffer.

6. The electrophoresis box according to claim 1, characterized in that: The oil is a liquid that phase separates from the gel.

7. The electrophoresis box according to claim 1, characterized in that: The oil is a non-polar solvent or a low-polar solvent.

8. The electrophoresis box according to claim 1, characterized in that: The oil is a lipid.

9. An electrophoresis method, characterized in that: include: preparing an electrophoresis box, the electrophoresis box having a gel tank for storing a gel and a buffer tank for injecting a buffer, and pre-packaging the gel and oil in contact with the gel, the buffer tank being a tank continuous with the gel tank, the buffer being injected into a sample to be electrophoresed in the gel; Adding the buffer solution into the buffer solution tank of the electrophoresis box to separate the oil from the gel; injecting the sample into the buffer solution put into the buffer solution tank; as well as An electric field is applied to the sample to cause electrophoresis of the sample in the gel.

Images