Nucleic acid electrophoresis gel

Abstract

The present invention relates to a nucleic acid electrophoresis test gel, which comprises a liquid agar gel and a solution of the acrylamide methylenebisacrylamide (Acr-Bis). By combining the liquid agar gel with the Acr-Bis solution, the present invention can improve the separation resolution of a gel formed from agar during nucleic acid electrophoresis, thereby making the properties of the nucleic acid electrophoresis test gel similar to those of commercially available agarose gel, and achieving good separation resolution for DNA fragments to be subjected to gel electrophoresis during nucleic acid electrophoresis tests.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid electrophoresis, and specifically relates to a gel for nucleic acid electrophoresis tests.

Background Art

[0002] Agarose is a chain-like neutral polysaccharide mainly composed of alternately linked D-galactose and 3,6-anhydro-L-galactose. Since it has a low content of charged groups such as sulfate and pyruvic acid, it has low electroosmosis, no adsorption to proteins and basic dyes, and can be used as an ideal electrophoresis medium. It is usually used in nucleic acid electrophoresis tests.

[0003] Agarose-related products are mainly used in the fields of medicine, scientific research, and biomedicine, and are closely related to human health. Currently in China, the production cost of agarose is high and the product quality is lower compared to imported products. Therefore, most of the raw materials and related products of agarose rely on imports and are very expensive. Therefore, there is an urgent need to develop new products that can replace commercially available agarose electrophoresis media.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The present invention aims to at least partly solve one of the technical problems in the related art. Therefore, one object of the present invention is to provide a gel for nucleic acid electrophoresis tests. The gel for nucleic acid electrophoresis tests produced by the present invention has good separation resolution for DNA fragments to be subjected to gel electrophoresis.

Means for Solving the Problems

[0005] In view of the above, according to an embodiment of the present invention, the present invention proposes a gel for nucleic acid electrophoresis tests, which includes an agar gel and a 0.25% to 2% by mass solution of methylene bisacrylamide Acr-Bis which is acrylamide.

[0006] The nucleic acid electrophoresis test gel according to the embodiment of the present invention achieves improved separation resolution during nucleic acid electrophoresis of gels formed with agar by combining an agar gel with methylenebisacrylamide (Acr-Bis), which is an acrylamide. As a result, the properties of the nucleic acid electrophoresis test gel become similar to those of commercially available agarose gels, and good separation resolution for DNA fragments targeted by gel electrophoresis is achieved when performing nucleic acid electrophoresis tests.

[0007] Furthermore, the nucleic acid electrophoresis test gel proposed in the above-described examples of the present invention may have the following additional technical features.

[0008] Optionally, the agar gel may be prepared by weighing 1.50 g of agar strip, adding it to distilled water so that the ratio of material to liquid is 1:100 w / v, mixing it uniformly, and treating it in a water bath at 100°C for 5 to 10 minutes.

[0009] Optionally, the Acr-Bis solution may be prepared by adding distilled water, buffer solution, 0.1 mL of 10% ammonium persulfate solution, and 0.004 mL of TEMED to 30% Acr-Bis (29:1) and mixing them uniformly.

[0010] Furthermore, the buffer solution is a 1.5 M Tris base solution containing 0.025% to 0.2% SDS, with a pH of 8.0 to 9.0.

[0011] Optionally, the ratio of the agar gel liquid to the Acr-Bis solution is between 5:1 and 40:1.

[0012] Additional aspects and advantages of the present invention are, in part, shown in the following description, in part, apparent from the following description, or understood through the practice of the present invention. [Brief explanation of the drawing]

[0013] [Figure 1]The dissolution temperatures of nucleic acid electrophoresis gel, commercially available agar, and commercially available agarose according to the embodiments of the present invention are shown. [Figure 2] The melting temperatures of nucleic acid electrophoresis gel, commercially available agar, and commercially available agarose according to the embodiments of the present invention are shown. [Figure 3] The solidification temperatures of the nucleic acid electrophoresis test gel, commercially available agar, and commercially available agarose according to the embodiments of the present invention. [Figure 4] The gel strengths of nucleic acid electrophoresis gels, commercially available agar, and commercially available agarose according to the embodiments of the present invention are shown. [Figure 5] This shows the transparency of nucleic acid electrophoresis gel, commercially available agar, and commercially available agarose according to the embodiments of the present invention. [Figure 6] The viscosity of nucleic acid electrophoresis gel, commercially available agar, and commercially available agarose according to the examples of the present invention. [Figure 7] The whiteness of nucleic acid electrophoresis gel, commercially available agar, and commercially available agarose according to the examples of the present invention. [Figure 8] These are gel electrophoresis diagrams of nucleic acid electrophoresis gels, commercially available agar, and commercially available agarose according to examples of the present invention. [Modes for carrying out the invention]

[0014] The technical solutions of the present invention will be described below with specific examples. It should be understood that one or more method steps described in the present invention do not preclude the existence of other method steps before or after the combination of such steps, or that other method steps may be inserted between these explicitly described steps. It should also be understood that these examples are merely illustrative of the present invention and do not limit its scope. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient means of identifying each method step and does not restrict the order of the method steps or limit the scope of the invention. Changes or adjustments in their relative relationships should be considered within the scope of the invention if they do not substantially alter the technical content.

[0015] To better understand the technical solutions described above, exemplary embodiments of the present invention will be described in more detail below. Although exemplary embodiments of the present invention have been shown, it should be understood that the present invention can be realized in various forms, without being limited to the embodiments described herein. Rather, these embodiments are provided to allow for a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0016] All of the test materials used in this invention are common, commercially available products and can all be purchased on the market.

[0017] The present invention will be described below with reference to specific examples. These examples are merely illustrative and do not limit the present invention in any way. [Examples]

[0018] Preparation of agar gel: Weigh 1.50 g of agar strip, add it to distilled water so that the ratio of material to liquid is 1:100 (w / v), mix thoroughly, heat the mixture in a water bath pot, stir evenly with a glass rod, set the water bath temperature to 100°C, and time for 5 minutes. Once the time is up, a uniformly dissolved agar gel is obtained.

[0019] Preparation of Acr-Bis solution: Weigh 4.1 mL of distilled water using a pipette gun and put it into a beaker. Next, weigh 3.3 mL of 30% Acr-Bis (29:1) and put it into the beaker. Further, weigh 2.5 mL of buffer solution (1.5 M Tris base, 0.1% SDS, adjusted to pH 8.8 with HCl) and put it into the beaker. Then, add 0.1 mL of 10% ammonium persulfate solution, and finally, add 0.004 mL of TEMED. After that, mix the solution uniformly.

Example

[0020] Add 4 mL of Acr-Bis solution to the agar gel (16 mL) obtained in Example 1 in proportion to prevent the coagulation of the Acr-Bis solution and obtain a gel for nucleic acid electrophoresis test.

[0021] Taking the above gel for nucleic acid electrophoresis test (modified agar gel), untreated commercial agar powder (manufacturer: Fujian Yange Marine Biotechnology Co., Ltd.) and commercial agarose (manufacturer: Shanghai Beijin Biotechnology Co., Ltd.) as comparisons, measure the nucleic acid electrophoresis resolution and physical and chemical properties (gel strength, turbidity, viscosity) of the above gel for nucleic acid electrophoresis test, commercial agar powder, and commercial agarose respectively.

[0022] Dissolution temperature: Put 0.1 g each of the samples of commercial agar powder, commercial agarose, and gel for nucleic acid electrophoresis test into test tubes, add 10 mL of distilled water, then stopper the test tubes and place them in a water bath at 30°C. Increase the temperature at a rate of 1°C / 15 min and observe the dissolution of the samples. The temperature when the solution in the test tube becomes clear and transparent without forming layers is taken as the dissolution temperature of the agar.

[0023] As shown in the results of Figure 1, the dissolution temperatures of commercial agarose, commercial agar powder, and the gel for nucleic acid electrophoresis test are 80.10°C, 88.64°C, and 89.79°C respectively.

[0024] Melting temperature: A 1.5% by mass agar solution was prepared, heated and dissolved, and 15 mL was taken and poured into a test tube with an inner diameter of 22 mm. A thermometer was inserted so that the mercury bulb was below the liquid surface, and the test tube was placed horizontally to allow it to solidify. The next day, one 3 mm diameter stainless steel bead was placed on the surface of the gel, and the test tube was heated in a water bath, gradually increasing the temperature of the gel. The temperature at which the stainless steel bead rapidly fell to the bottom of the test tube was observed and defined as the melting temperature.

[0025] As shown in Figure 2, the melting temperatures of commercially available agarose, commercially available agar powder, and nucleic acid electrophoresis gel were 90.77°C, 94.36°C, and 91.67°C, respectively.

[0026] Solidification temperature: Prepare a 1.5% by mass agar solution, heat and dissolve it, then take 10 mL and pour it into a test tube with an inner diameter of 15 mm. Insert a thermometer so that the mercury bulb is below the liquid surface. Slowly lower the temperature of the liquid gel (approximately 1°C / min) until the test tube is tilted 90°. The temperature at which the surface of the liquid solidifies and stops flowing is defined as the solidification temperature.

[0027] As shown in Figure 3, the solidification temperatures of commercially available agarose, commercially available agar powder, and nucleic acid electrophoresis gel were 37.17°C, 44°C, and 38.7°C, respectively.

[0028] Gel Strength: The gel strength of the three agar powder samples was measured according to the national standard GB 1975-2010 Agar Gel Strength Measurement Method.

[0029] As shown in Figure 4, the gel strengths of commercially available agarose, commercially available agar powder, and nucleic acid electrophoresis gel were 2232 g / cm², respectively. 2 1189g / cm³ 2 , and 1075g / cm³ 2 That is the case.

[0030] Transparency: Prepare a 1% by mass agar solution, pour it into a cuvette while still hot, leave it at room temperature for 24 hours, scan it between 400 and 800 nm using a UV spectrophotometer, determine the maximum absorption wavelength at 700 nm, and measure the transmittance (T) of the sample at the maximum absorption wavelength.

[0031] As shown in Figure 5, the transparency of commercially available agarose, commercially available agar powder, and nucleic acid electrophoresis gels was 98.9%, 92.4%, and 84.7%, respectively.

[0032] A 1.5% by mass agar solution is prepared, microwaved until the solution is uniform and clear, poured into a 300 mL tall beaker, and treated in a 60°C constant temperature water bath for 30 minutes. After the temperature stabilizes, the viscosity is measured using a DV-C digital viscometer.

[0033] As shown in Figure 6, the viscosity of commercially available agarose is 4.28 mPa·s, the viscosity of commercially available agar powder is 7.38 mPa·s, and the viscosity of the nucleic acid electrophoresis gel is 5.53 mPa·s. Compared to commercially available agar powder, the viscosity of the nucleic acid electrophoresis gel is significantly lower, indicating an improvement in the quality of the agar powder.

[0034] Whiteness: The whiteness of the three agar powder samples was measured according to the national standard GB12097-1989 Starch Whiteness Measurement Method.

[0035] As shown in Figure 7, the whiteness of the nucleic acid electrophoresis gel (68.8%) was lower than that of commercially available agarose (85.59%), but significantly higher than that of commercially available agar powder (66.05%), indicating that the whiteness of the nucleic acid electrophoresis gel had improved to some extent.

[0036] Nucleic acid electrophoresis: The above nucleic acid electrophoresis test gel, untreated commercial agar powder, and commercial agarose were poured into an Erlenmeyer flask. The Erlenmeyer flask was placed in a microwave oven, and the power and time (1180W, 1-2 min) were set according to the gel concentration and buffer volume to ensure that the gel was completely dissolved. Once the temperature of the gel in the Erlenmeyer flask had dropped to about 60°C, nucleic acid fuel (EB ethidium bromide) was added. The liquid gel was poured into a gel tray, and the comb was inserted in the corresponding position. After standing at room temperature for at least 30 minutes, the comb was removed. The prepared gel, along with the gel tray, was placed in an electrophoresis tank, electrophoresis buffer was poured in, the marker and the nucleic acid to be analyzed were added to the comb holes, the electrophoresis conditions were set, and electrophoresis was started. After electrophoresis was completed, the results were analyzed using a gel imaging system or UV spectrophotometer.

[0037] A 1.5% gel was prepared using the nucleic acid electrophoresis gel described above. A 5000 bp DNA molecule standard was used, and the gel was transferred at 120 V for 33 minutes. As a control experiment, 1.5% gels were prepared using untreated commercial agar powder and commercial agarose, and electrophoresis was performed. As shown in Figure 1, A is an image of the commercial agarose gel, B is an image of the commercial agar powder gel, C is an image of the 0.25% polyacrylamide + agar gel, D is an image of the 0.5% polyacrylamide + agar gel, E is an image of the 1% polyacrylamide + agar gel, and F is an image of the 2% polyacrylamide + agar gel. In the figure, the separation effect of the commercial agarose gel for nucleic acid fragments was high, with a dark background color, slight impurities, relatively bright bands, and high resolution. The separation effect of the commercial agar gel for nucleic acid fragments was low, with a dark background color, impurities, relatively dark bands, and low resolution. Compared to commercially available agarose gels, the 0.5% nucleic acid electrophoresis gel was able to separate larger nucleic acid molecular fragments more thoroughly, exhibiting a darker background, minimal impurities, narrower and brighter bands, and higher resolution.

[0038] As described above, the analysis of the physical and chemical properties of the nucleic acid electrophoresis gel showed that its dissolution temperature, gelation temperature, and melting temperature were all superior to those of commercially available agarose, and its viscosity and whiteness were both superior to those of commercially available agar powder, and were close to those of commercially available agarose.

[0039] In this specification, any reference to terms such as “one embodiment,” “several embodiments,” “example,” “specific example,” or “several examples” means that the specific features, structures, materials, or properties described in relation to that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, a general description of the above terms does not necessarily mean the same embodiment or example. Furthermore, the specific features, structures, materials, or properties described may be combined in an appropriate manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine or combine different embodiments or examples described herein.

[0040] Although the above examples have been described, these examples are illustrative and should not be understood as limiting the present invention. Those skilled in the art can modify, alter, substitute, and transform the above examples within the scope of the present invention.

Claims

1. A gel for nucleic acid electrophoresis testing, characterized by containing an agar gel and 0.25% to 2% by mass of polyacrylamide crosslinked with methylenebisacrylamide.

2. A method for producing a nucleic acid electrophoresis test gel, comprising the steps of: heating and dissolving agar to obtain an agar gel liquid; preparing an Acr-Bis mixed solution by adding a buffer, ammonium persulfate solution, and TEMED to an aqueous solution of acrylamide and methylenebisacrylamide; and before the Acr-Bis mixed solution solidifies, adding the Acr-Bis mixed solution to the agar gel liquid and mixing to obtain a nucleic acid electrophoresis test gel.

3. The method for producing a nucleic acid electrophoresis test gel according to claim 2, wherein the step of obtaining the agar gel liquid is to add agar to distilled water so that the ratio of material to liquid is 1:100 w / v, mix uniformly, and prepare by treating in a water bath at 100°C for 5 to 10 min.

4. The method for producing a nucleic acid electrophoresis gel according to claim 2 or 3, wherein the buffer solution is a 1.5 M Trisbase solution containing 0.025% to 0.2% SDS, and has a pH of 8.0 to 9.

0.

5. The method for producing a nucleic acid electrophoresis gel according to claim 2 or 3, wherein the ratio of the agar gel liquid to the Acr-Bis mixed solution is 5:1 to 40:1.

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