Electrophoresis cassette and electrophoresis method

Abstract

With the present invention it is possible to form wells without a comb and to use a gel having high degrees of convenience and freedom, and a decline in sample separation performance due to gel swelling is prevented. This electrophoresis cassette 1 comprises: a gel tank 2; a gel 7 stored in the gel tank 2; a buffer liquid tank 3 connected to the gel tank 2 and filled with a buffer liquid into which a sample (target biological substance) to undergo electrophoresis in the gel 7 is loaded; and an oil 8 in contact with the gel 7 in the gel tank 2. Additionally, the gel 7 and the oil 8 are prepackaged in the electrophoresis cassette 1.

Description

Technical Field

[0001] The present disclosure relates to an electrophoresis cassette and an electrophoresis method, and more particularly to an electrophoresis cassette in which a gel is prepackaged and an electrophoresis method using the electrophoresis cassette.

Background Art

[0002] A gel electrophoresis method for analyzing biological substances such as nucleic acids and proteins is known, which utilizes the phenomenon that when an electric field is applied to a charged substance, the substance moves in the direction of the electrode of the opposite polarity. Generally, electrophoresis gels such as agarose gels and acrylamide gels are used as supports for biological substances. Since the migration speed in the electrophoresis gel varies depending on the molecular weight of the biological substance, the biological substance is separated as different bands for each molecular weight. The gel electrophoresis method has a high resolution for separating biological substances, and is therefore also used for measuring the fragment length of DNA to understand the state of a sample. In addition, it is also used for quantifying DNA of a specific length by fluorescently labeling the DNA and observing its brightness.

[0003] Normally, an electrophoresis gel is adjusted and used each time immediately before electrophoresis. However, the preparation of an electrophoresis gel has a problem of being laborious because gel dissolution is required for an agarose gel and reagent adjustment and degassing are required for a polyacrylamide gel.

[0004] As a prepackaged polyacrylamide gel that does not require adjustment of an electrophoresis gel and can be electrophoresed immediately, for example, Patent Documents 1 and 2 disclose a method of preparing a gel between glass plates called a slab gel and storing them using a buffer solution of low pH.

[0005] In addition, Patent Document 3 discloses an electrophoresis apparatus in which a fluorescent dye is added to a prepackaged gel that has been prepared in advance, and fluorescence staining of a sample is unnecessary.

[0006] Furthermore, Patent Document 4 discloses an electrophoresis cassette in which the gel and buffer are pre-packaged. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent No. 7422670 [Patent Document 2] U.S. Patent No. 6,783,651 [Patent Document 3] U.S. Patent No. 8562802 [Patent Document 4] U.S. Patent No. 8591713 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] However, pre-packaged gels require structures called wells to receive samples, and maintaining these structures presents a challenge due to constraints.

[0009] Many pre-packaged gels have a resin plate called a comb inserted into the gel. Before use, the comb is removed, and the sample is poured into the well formed therein (Patent Documents 1, 2, and 3). When using cassettes containing these pre-packaged gels in an automated electrophoresis system, a comb removal mechanism must be installed. Furthermore, if an operator manually removes the comb before using the automated electrophoresis system, the wells are exposed within the system for a certain period of time before electrophoresis begins, which can lead to contamination and drying of the buffer solution.

[0010] Patent Document 4 discloses an electrophoresis cassette that pre-packages the buffer solution in contact with the gel and holds the wells without a comb, but the gel composition is limited to cross-linked gels to prevent the gel from swelling during storage. Furthermore, it is necessary to select a composition that swells less even among cross-linked gels. In addition, the salt concentrations of the buffer solution and the gel must be matched to prevent swelling and shrinkage due to osmotic pressure differences.

[0011] Therefore, the present disclosure aims to provide an electrophoresis cassette and electrophoresis method that enable the formation of wells without a comb, allow the use of convenient and flexible gels, and prevent a decrease in sample separation performance due to gel swelling. [Means for solving the problem]

[0012] To solve the above problems, the electrophoresis cassette of this disclosure comprises a gel tank for containing a gel, and a tank continuous with the gel tank, into which a buffer solution is introduced into which a sample to be electrophoresed in the gel is injected, and the gel and the oil in contact with the gel are pre-packaged.

[0013] Furthermore, in order to solve the above problems, the electrophoresis method of this disclosure comprises preparing an electrophoresis cassette in which the gel and oil in contact with the gel are pre-packaged, the gel being contained in a gel tank, and a buffer tank continuous with the gel tank into which a buffer is introduced into which a sample to be electrophoresed in the gel is injected; introducing a buffer into the buffer tank of the electrophoresis cassette to separate the oil and the gel; injecting the sample into the buffer introduced into the buffer tank; and applying an electric field to the sample to electrophorese the sample in the gel. [Effects of the Invention]

[0014] According to the electrophoresis cassette and electrophoresis method described herein, wells can be formed without a comb, a convenient and flexible gel can be used, and a decrease in sample separation performance due to gel swelling can be prevented. [Brief explanation of the drawing]

[0015] [Figure 1] It is a front view of the electrophoresis cassette according to the embodiment. [Figure 2] It is a cross-sectional view of the electrophoresis cassette according to the embodiment. [Figure 3] It is a graph explaining the result of the gel swelling experiment when the buffer solution according to the comparative example is overlaid. [Figure 4] It is a graph explaining the result of the gel swelling experiment when the oil according to Example 1 is overlaid. [Figure 5] It is a diagram showing the electrophoresis image according to Example 2.

Mode for Carrying Out the Invention

[0016] In all the drawings for explaining this embodiment, those having the same function are given the same reference numerals, and the repeated description may be omitted. Further, the present invention is not construed as being limited to the description of the embodiments shown below. The present invention is defined by the appended claims, but it will be easily understood by those skilled in the art that the specific configuration can be changed without departing from the spirit or gist thereof.

[0017] The positions, sizes, shapes, ranges, etc. of the respective components shown in the drawings and the like may not represent the actual positions, sizes, shapes, ranges, etc. in order to facilitate the understanding of the invention. For this reason, the present invention is not necessarily limited to the positions, sizes, shapes, ranges, etc. disclosed in the drawings and the like. ​​​​The electrophoresis cassette 1 according to an embodiment of the present disclosure will be described with reference to FIGS. 1 and 2. FIGS. 1 and 2 are diagrams showing the configuration of the electrophoresis cassette 1 according to the embodiment. In the example of FIG. 2, a support 9 covering the gel 7 is provided. The support 9 is electrically resistive (including those that are insulating and those with high electrical resistance (not only perfect insulators but also those that conduct some electricity)).

[0019] The electrophoresis cassette 1 includes an upper electrode 4a and a lower electrode 4b for applying a voltage. Although not shown, a power source is connected to the upper electrode 4a and the lower electrode 4b, and the configuration is such 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 be connected to the power source.

[0020] Hereinafter, the case where the sample (target biological substance) to be recovered is nucleic acid will be described as an example.

[0021] The gel 7 is a separation medium used for separating the target biological substance and unwanted substances. As the gel 7, for example, a known one such as an agarose gel or a polyacrylamide gel can be used. Although there is no particular limitation on the thickness of the gel 7, from the viewpoint that the band of the biological substance obtained by electrophoresis is sharp and easy to visually recognize, it is preferably 0.5 to 18 mm. Note that the thickness of the gel 7 does not have to be constant.

[0022] The electrophoresis cassette 1 includes a gel tank 2 capable of accommodating the gel 7. The gel tank 2 is provided with an injection port 5 for injecting the gel 7. The gel 7 is pre-packaged in the gel tank 2.

[0023] The electrophoresis cassette 1 includes a buffer tank 3 capable of accommodating a buffer solution into which a sample (target biological substance) that electrophoreses in the gel 7 is injected. The buffer tank 3 is a tank continuous with the gel tank 2. The internal space of the buffer tank 3 and the internal space of the gel tank 2 are spatially continuously connected. Immediately before electrophoresis, the buffer solution is introduced into the buffer tank 3. The buffer solution is not pre-packaged.

[0024] The gel 7 contained in the gel tank 2 is in contact with the oil 8. This oil 8 is pre-packaged in the buffer tank 3. The oil 8 is a liquid that undergoes phase separation with water or gel 7, and can be, for example, a non-polar solvent, a low-polarity solvent, or a lipid. The oil 8 contains benzene or toluene. The interfacial tension between the oil 8 and water or gel 7 is such that the phase separation state is maintained, and can be, for example, 10 mN / m or more. The surface tension of the oil 8 can also be, for example, 10 mN / m. Furthermore, the interfacial tension between water or gel 7 and oil 8 can be less than the surface tension of the oil 8.

[0025] Oil 8 has a lower specific gravity than the buffer solution. This makes it easier for an interface to form between the buffer solution and gel 7 during electrophoresis.

[0026] The support 9 is formed using resin or the like. By providing an opening in the buffer tank 3 or a separate piercing mechanism, it becomes possible to inject a sample containing the target biological substance to be separated into the buffer tank 3. In this embodiment, an opening for introducing the sample toward the top surface is formed at one end of the buffer tank 3 or near it.

[0027] The target biomaterial is injected into buffer tank 3 as an injection solution mixed with a liquid having a specific gravity greater than that of the buffer. Examples of solvents to which the target biomaterial is mixed include aqueous glycerol solution and sugar water. When the solvent is aqueous glycerol solution, the glycerol concentration can be, for example, 6%. The viscosity of the injection solution can be, for example, 1 mPa·s.

[0028] The distance between the buffer reservoir 3 and the lower electrode 4 can be set arbitrarily. This distance can be appropriately designed according to the composition of the gel 7 (e.g., gel concentration), the molecular weight of the target biomaterial, etc.

[0029] In this embodiment, the gel tank 2 and buffer tank 3 are substantially rectangular parallelepipeds, but their structure, shape, size, etc., are not limited to those shown. The structure, shape, size, etc., of the gel tank 2 and buffer tank 3 can be arbitrarily set. The widthwise dimensions of the gel tank 2 and buffer tank 3 (i.e., the horizontal dimensions perpendicular to the direction of the electric field; not shown in Figure 2) may be equal or different.

[0030] In this embodiment, the electrophoretic direction of the biomaterial is the vertical direction (Z direction in the figure), and the gel tank 2 and buffer tank 3 are arranged continuously in the vertical direction; however, the electrophoretic direction is not limited to the vertical direction.

[0031] (Electrophoresis method) Next, we will explain the electrophoresis method using the electrophoresis cassette 1 described above.

[0032] First, prepare the electrophoresis cassette 1. The electrophoresis cassette 1 comprises a gel tank 2, a gel 7 contained within the gel tank 2, a buffer tank 3 continuous with the gel tank 2 into which the buffer solution into which the sample to be electrophoresed in the gel 7 is injected is added, and an oil 8 in contact with the gel 7 in the gel tank 2. The gel 7 and oil 8 are pre-packaged in the electrophoresis cassette 1.

[0033] Next, buffer solution is added to the buffer tank 3 of the electrophoresis cassette 1 to separate the oil 8 and gel 7.

[0034] Then, the sample is injected into the buffer solution placed in buffer tank 3.

[0035] Finally, an electric field is applied to the sample, and the sample is subjected to electrophoresis in gel 7.

[0036] (Comparative example) The following describes a comparative example.

[0037] A gel was formed by polymerizing acrylamide and a crosslinking agent at a total monomer content of 12% (w / v) in a 2 mL tube. Tris Glycine was used as the buffer solution. The crosslinking agents used were methylenebisacrylamide, dihydroethylenebisacrylamide, and bisacryloylcystamine in the weight ratios shown in Figure 3. Equal volumes of Tris Glycine buffer solution of the same concentration were injected onto the gel.

[0038] The buffer solution was decanted and removed every few days, and the weight of the gel was measured. After measurement, the decanted buffer solution was returned to the original tube and stored again. This process was repeated for 38 days.

[0039] The results are shown in Figure 3. The graph plots the weight swelling rate over time (with the initial value set to 100%). It can be seen that the amount of swelling increases when the ratio of crosslinking agent is low.

[0040] (Example 1) Next, Example 1 will be described.

[0041] After preparing the gel layer in the same manner as in the comparative example, the weight swelling rate was evaluated by pouring oil or buffer into the tube. The gel was prepared with a total monomer content of 12% (w / v) of acrylamide and crosslinking agent, using dihydroethylenebisacrylamide as the crosslinking agent at a weight ratio of 149:1 to acrylamide. The buffer used was Tris Glycine prepared to pH 9.5 or Bis-Tris Tricine BES prepared to pH 6.9. Mineral oil, silicone oil, or toluene were used as the oil.

[0042] The results are shown in Figure 4. When buffer solution was injected into the tubes, swelling of 15% to 35% occurred in 6 days, but when oil was injected into the tubes, the swelling rate was less than 5% in 6 days.

[0043] (Example 2) Next, Example 2 will be described.

[0044] A gel was formed by polymerizing acrylamide and a crosslinking agent in a support with a channel structure formed on a PET sheet, using a total monomer content of 12% (w / v). Dihydroethylene bisacrylamide was used as the crosslinking agent at a concentration of 6% (w / w) of acrylamide. Tris Glycine was used as the buffer. Buffer or oil was injected into a buffer bath on the gel, and the gel was stored at 37°C for 4 days before electrophoresis. A 100 bp ladder was used for the electrophoresis samples.

[0045] The electrophoretic patterns are shown in Figure 5. When the buffer solution was injected 4 days ago (4 days, Bubbfer), the gel pores were larger and the electrophoretic movement was faster compared to immediately after the buffer solution was injected (0 days, Bubbfer). Conversely, when the oil solution was injected 4 days ago (4 days, Mineral oil and Silicon oil), the electrophoretic state was similar to that of immediately after the buffer solution was injected (0 days, Bubbfer).

[0046] (Effects of the embodiment) In this embodiment, the gel 7 is pre-packaged in the gel tank 2, and the oil 8 that comes into contact with the gel 7 in the gel tank 2 is also pre-packaged, allowing for the formation of wells without a comb. This eliminates the need for a comb removal mechanism. Furthermore, since the gel 7 and oil 8 are pre-packaged, contamination and drying of the buffer solution can be prevented.

[0047] Furthermore, in this embodiment, the composition of gel 7 to prevent swelling of gel 7 is not limited, so a gel 7 with high convenience and flexibility can be used.

[0048] Furthermore, in this embodiment, by providing oil 8 in contact with gel 7, it is possible to prevent a decrease in sample separation performance due to swelling of gel 7.

[0049] Furthermore, in this embodiment, it is necessary to inject the buffer solution immediately before electrophoresis, but this can be easily done as it is the same dispensing operation as the injection solution (a mixture of sample and solvent).

[0050] Furthermore, in this embodiment, since the oil 8 has a lower specific gravity than the buffer solution, when the electrophoresis cassette 1 is used, the buffer solution is injected into the buffer solution tank 3, which replaces the oil 8 on the upper side, creating a gel 7-buffer solution interface and enabling electrophoresis.

[0051] Furthermore, in this embodiment, since the buffer solution is injected immediately before electrophoresis, it is possible to significantly change the salt concentration between the gel 7 and the buffer solution. This allows for band concentration and improved separation performance due to the difference in conductivity between the buffer solution and the gel 7.

[0052] This disclosure is not limited to the embodiments described above, and includes various modifications. For example, the embodiments described above are described in detail for the purpose of explaining this disclosure clearly, and are not necessarily limited to those having all the configurations described. In addition, it is possible to add, delete, or replace some of the configurations of each embodiment with other configurations. [Explanation of Symbols]

[0053] 1… Electrophoresis cassette 2…Gel cell 3…Buffer tank 4a...Electrode 4b...Electrode 5…Injection port 7…Gel 8… Oil 9...Support

Claims

1. A gel tank for containing the gel, The system comprises a tank continuous with the gel tank, into which a buffer solution is introduced into which a sample to be electrophoresed in the gel is injected, The gel and the oil in contact with the gel are pre-packaged. An electrophoresis cassette characterized by the following features.

2. The interfacial tension between the gel and the oil is 10 mN / m or more. The electrophoresis cassette according to feature 1.

3. The surface tension of the oil is 10 mN / m or more. The electrophoresis cassette according to feature 1.

4. The interfacial tension between the gel and the oil is less than the surface tension of the oil. The electrophoresis cassette according to feature 1.

5. The specific gravity of the oil is less than that of the buffer solution. The electrophoresis cassette according to feature 1.

6. The oil is a liquid that undergoes phase separation from the gel. The electrophoresis cassette according to feature 1.

7. The oil is a nonpolar solvent or a low-polarity solvent. The electrophoresis cassette according to feature 1.

8. The oil is a lipid. The electrophoresis cassette according to feature 1.

9. The electrophoresis cassette is prepared by pre-packaging the gel and the oil in contact with the gel, comprising a gel tank for containing the gel, and a buffer tank continuous with the gel tank into which a buffer solution is introduced into which a sample to be electrophoresed in the gel is injected. The buffer solution is added to the buffer tank of the electrophoresis cassette to separate the oil and the gel. Injecting the sample into the buffer solution placed in the buffer solution tank, and The process involves applying an electric field to the sample and performing electrophoresis on the sample in the gel. An electrophoresis method characterized by the following features.

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