Method for preparing a high-purity DNA fragment mixture

Abstract

The present invention relates to a method for preparing a highly pure DNA fragment mixture using isopropanol and magnesium chloride. The isopropanol can be used to effectively remove impurities such as water, blood, and fat, thereby increasing the purity of the DNA fragment mixture. The magnesium chloride can also be used to prepare a DNA fragment mixture with a relatively narrow molecular weight range of 1,000 kDa to 10,000 kDa.

Description

Technical Field

[0001] Background of the Invention 1. Field of the Invention The present invention relates to a method for producing a mixture of DNA fragments, specifically, a method for preparing a high-purity mixture of DNA fragments using isopropanol and magnesium chloride.

Background Art

[0002] 2. Description of Related Art Nucleotides are structural units that make up nucleic acids such as DNA and RNA. A polymer consisting of 10 or more nucleotides is called a polynucleotide (PN). In addition, a nucleotide is composed of a combination of one 5-carbon sugar (pentose), one phosphate, and one base (adenine, guanine, thymine, or cytosine). The balance of the combinations that make up the nucleotides of masu salmon and sockeye salmon is 96.5% identical to that of humans. This high similarity is important in that all nucleic acids can be used without being discarded.

[0003] On the other hand, a DNA fragment mixture is a mixture in which DNA exists in the form of fragments with reduced molecular weight. Since the DNA fragment mixture contains essential components of cells, it is used for various purposes such as medical devices and pharmaceuticals for the treatment and improvement of wounds, by injecting it into wounds, musculoskeletal regions, or joints, knees, and joint cavities to relieve mechanical friction and pain, or for cosmetics, food additives, and biochemical experimental materials for the purpose of improving wrinkles related to cell activity, etc., and its value is increasing.

[0004] As a conventional method for preparing DNA fragment mixtures, Korean Patent Publication No. 10-390529 discloses a method for extracting nucleic acids using an ultrafiltration membrane. However, this method has the disadvantage of being unable to effectively remove impurities such as RNA, proteins, fats, and water, making it difficult to extract high-purity nucleic acids. In addition, Korean Patent Publication No. 10-2019-0065676 discloses a method for reducing the molecular weight of DNA by adjusting the pH, but this method has the disadvantage of being difficult and dangerous because it uses strong acids and strong bases.

[0005] Therefore, the inventors investigated a method for preparing a high-purity DNA fragment mixture that safely reduces the molecular weight of DNA and effectively removes impurities. They confirmed that by using isopropanol to remove impurities and magnesium chloride to mix the DNA fragments, the molecular weight of DNA can be safely reduced with high yield and high purity, thus completing the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0006] Summary of the Invention The object of the present invention is to provide a method for preparing a high-purity DNA fragment mixture. Another object of the present invention is to provide a high-purity DNA fragment mixture.

[0007] To achieve the above objective, the present invention provides a method for preparing a high-purity DNA fragment mixture comprising the following steps. (Step 1) Treat the milt with isopropanol to obtain milt from which impurities have been removed. (Step 2) The milt from which the above impurities have been removed is treated by adding it to an aqueous sodium chloride solution. (Step 3) Add an aqueous sodium lauryl sulfate solution to an aqueous sodium chloride solution containing fish milt to obtain a primary fish milt lysate solution in which the cells are broken down and nucleic acids are extracted. (Step 4) The primary milt lysate solution is precipitated with ethanol, and the precipitate is separated to obtain the secondary milt lysate solution. (Step 5) Add sodium chloride to the secondary albino lysate solution and first inactivate the virus at a high temperature of 80-100°C. (Step 6) Add magnesium chloride to the virus-inactivated alfonsino lysate solution to obtain a DNA fragment mixture with reduced nucleic acid molecular weight, and (Step 7) The DNA fragment mixture is treated with ethanol to reduce the nucleic acid molecular weight of the DNA fragments, thereby secondarily inactivating the virus, and then obtaining a precipitate.

[0008] In addition, the present invention provides a high-purity DNA fragment mixture prepared by the above method. [Effects of the Invention]

[0009] Favorable effects The method for preparing a high-purity DNA fragment mixture according to the present invention has the effect of effectively removing impurities such as water, blood, and fat by using isopropanol, thereby increasing the purity of the DNA fragment mixture, and the effect of preparing a DNA fragment mixture having a relatively narrow molecular weight range of 1,000 kDa to 10,000 kDa by using magnesium chloride. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a photograph showing a comparison of the transparency of DNA fragment mixtures prepared according to the pretreatment method. [Figure 2] Figure 2 is a photograph showing a comparison of the molecular weight reduction effects of nucleic acid molecular weight reduction methods. [Figure 3] Figure 3 is a photograph showing a comparison of the molecular weight reduction effect of magnesium chloride treatment time. [Figure 4]Figure 4 is a photograph showing a comparison of the molecular weights of Example 1 and the control group (PN, HTL Co.). [Figure 5A] Figure 5A is a graph showing a comparison of the GPC assay results for the control group. [Figure 5B] Figure 5B is a graph showing a comparison of the GPC assay results for Example 3. [Figure 5C] Figure 5C is a graph showing a comparison with the GPC assay results of Example 2. [Figure 5D] Figure 5D is a graph showing a comparison with the GPC assay results of Example 1. [Figure 6] Figure 6 is a graph showing a comparison of viscoelasticity between Example 1 and the control group. [Modes for carrying out the invention]

[0011] Description of Preferred Embodiments The present invention will be described in detail below. In one embodiment of the present invention, the present invention provides a method for preparing a high-purity DNA fragment mixture comprising the following steps. (Step 1) Treat the milt with isopropanol to obtain milt from which impurities have been removed. (Step 2) The milt from which the above impurities have been removed is treated by adding it to an aqueous sodium chloride solution. (Step 3) Add an aqueous sodium lauryl sulfate solution to an aqueous sodium chloride solution containing fish milt to obtain a primary fish milt lysate solution in which the cells are broken down and nucleic acids are extracted. (Step 4) The primary milt lysate solution is precipitated with ethanol, and the precipitate is separated to obtain the secondary milt lysate solution. (Step 5) Add sodium chloride to the secondary albino lysate solution and first inactivate the virus at a high temperature of 80-100°C. (Step 6) Add magnesium chloride to the virus-inactivated alfonsino lysate solution to obtain a DNA fragment mixture with reduced nucleic acid molecular weight, and (Step 7) By treating the DNA fragment mixture with ethanol to reduce the nucleic acid molecular weight of the DNA fragments, the virus is secondarily inactivated, and then a precipitate is obtained.

[0012] In the above step 1, isopropanol is added to the white fish sperm, the mixture of the white fish sperm and isopropanol is pulverized, and after leaving the mixture for 5 to 30 minutes, the impurity removal is achieved by separating the pulverized white fish sperm and isopropanol. The isopropanol can be added in an amount of 3 to 6 times, preferably 4 to 5 times, more preferably 5 times the weight of the white fish sperm. The white fish sperm may be masu white fish sperm or salmon white fish sperm, and preferably salmon white fish sperm.

[0013] The above steps 2 and 3 are steps for cell lysis and nucleic acid extraction. In the above step 2, the cells are lysed at a high temperature through a heat treatment method, and the nucleic acid is extracted using sodium chloride. Also, in step 3, sodium lauryl sulfate is used to lyse the cells that were not lysed in step 2 and further remove impurities such as fats and proteins.

[0014] More specifically, in the above step 2, an aqueous sodium chloride solution with a concentration of 20 to 50% by weight is added to the white fish sperm from which impurities have been removed, treated at a temperature of 90 to 100 °C for 1 to 4 hours, and then cooled to 60 to 65 °C. Preferably, an aqueous sodium chloride solution with a concentration of 25 to 35% by weight is added, followed by treatment at a temperature of 95 to 100 °C for 1 to 2 hours.

[0015] In the above step 3, an aqueous sodium lauryl sulfate solution is added to the aqueous sodium chloride solution containing white fish sperm at 10 to 20% by weight to a final concentration of 0.5 to 2% by weight, and treated at 10 to 30 °C for 20 to 40 minutes. Preferably, the aqueous sodium lauryl sulfate solution is added so that the final concentration becomes 1 to 1.5% by weight.

[0016] In step 4 above, the primary milt lysate solution is precipitated by adding ethanol to 50-80% of the final concentration, and the precipitate is separated to obtain the secondary milt lysate solution.

[0017] More specifically, the primary milt lysate solution is filtered through a filter with a pore size of 50-10 μm, ethanol is added to the filtrate to a final concentration of 50-80%, and the solution is left to stand for 20 minutes to separate the precipitate. Next, the precipitate is dissolved in distilled water to obtain the secondary milt lysate solution.

[0018] Step 5, the initial virus inactivation, involves adding sodium chloride to a final concentration of 0.5–2 M and treating at a high temperature of 80–100°C for 12–20 hours. Preferably, sodium chloride can be added to a final concentration of 0.7–1.5 M and treated at a high temperature of 80–90°C.

[0019] Step 6 involves adding magnesium chloride to bring the final concentration to 10-30 mM and processing at 80-100°C for 2-5 hours. Preferably, magnesium chloride can be added to bring the final concentration to 15-35 mM and the processing can be carried out at a high temperature of 80-90°C. In step 6 above, magnesium chloride breaks down nucleic acids, reducing the molecular weight of DNA fragments.

[0020] Step 7 involves adding 50-80% ethanol and treating for 12-20 hours. Specifically, in this step, ethanol is added to a final concentration of 50-80%, left to stand for 10-30 minutes to separate the precipitate, and then 50-80% ethanol is added to the precipitate and treated for 12-20 hours. Preferably, 60-75% ethanol is added and treated for 15-16 hours.

[0021] In another aspect of the present invention, the present invention provides a high-purity DNA fragment mixture prepared by the above method. The DNA fragment is characterized by having a molecular weight of 1,000 to 10,000 kDa. In the present invention, "DNA fragment mixture" means PN (polynucleotide) or PDRN (polydeoxyribonucleotide), and preferably means PN.

[0022] In this invention, "impurities" include blood, water, protein, fat, endotoxins, etc., which are eluted by the decomposition of the cells of the fish milt. In this invention, "processing" means immersion or stirring.

[0023] The present invention will be described in detail below with reference to the following examples and experimental examples. However, the following examples and experimental examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

[0024] Example 1: Preparation of high-purity DNA fragment mixture 1 A mixture of high-purity DNA fragments was prepared according to the following procedure. (Step 1) After thawing 200g of frozen salmon milt, internal organs such as blood vessels, blood, and eggs were removed. Isopropanol equivalent to five times the weight of the milt was added to the milt, and it was ground in a blender and left to stand for 10 minutes. Then, the milt powder and isopropanol were separated by centrifugation and filtration. (Step 2) Sterile distilled water was added to the pulverized material from Step 1 and stirred, then 30% sodium chloride was added and the mixture was slowly stirred at 95°C for 1 hour. The reaction mixture was then cooled to 60-65°C.

[0025] (Step 3) Sodium lauryl sulfate was added to the aqueous solution of sodium chloride containing the fish milt to a final concentration of 1%, and the mixture was stirred slowly for 30 minutes. (Step 4) The lysate solution from Step 3 was filtered through a filter with a pore size of 50-10 μm. Ethanol was then added to the filtrate to a final concentration of 50-80%, and the mixture was left to stand for 20 minutes. After standing, the resulting precipitate was washed by adding 50-80% ethanol and stirring slowly. After washing, the precipitate was dehydrated with 95% ethanol and dried using a hot air dryer (55°C) or a vacuum dryer (45°C, -0.09 to -0.1 MPa). The dried product was then completely dissolved in sterile distilled water to a final concentration of 1-3%.

[0026] (Step 5) The lysate solution from Step 4 was filtered through a 1-0.5 μm filter, sodium chloride was added to it to a final concentration of 1 M, and then the mixture was slowly stirred at 85°C for 16 hours. (Step 6) Magnesium chloride was added to the lysate solution from Step 5 to a final concentration of 20 mM, and the mixture was slowly stirred at 85°C for 3 hours and 30 minutes.

[0027] (Step 7) The lysate solution from Step 6 was sterile filtered through a 0.22 μm pore size filter. Ethanol was added to the filtrate to a final concentration of 70%, and it was left to stand for 20 minutes to obtain a precipitate. The precipitate was washed with 70% ethanol and stirred slowly for 16 hours. (Step 8) After adding 95% ethanol to the precipitate washed in Step 7, the mixture was slowly stirred for 10 minutes to dehydrate it, and then dried in a hot air dryer (55°C) or vacuum dryer (45°C, -0.09 to -0.1 MPa) to prepare a high-purity DNA fragment mixture with a molecular weight of 1,000 to 10,000 kDa.

[0028] Example 2: Preparation of a high-purity DNA fragment mixture 2 A high-purity DNA fragment mixture was prepared in the same manner as in Example 1, except that the magnesium chloride treatment in (Step 6) of Example 1 was carried out at 85°C for 2 hours.

[0029] Example 3: Preparation of a high-purity DNA fragment mixture 3 A high-purity DNA fragment mixture was prepared in the same manner as in Example 1, except that the magnesium chloride treatment in (Step 6) of Example 1 was carried out at 85°C for 1 hour.

[0030] Comparative Example 1 The DNA fragment mixture was prepared in the same manner as in Example 1, except that distilled water was added instead of isopropanol in (Step 1) of Example 1.

[0031] Comparative Example 2 The DNA fragment mixture was prepared in the same manner as in Example 1, except that isopropanol was not added in (Step 1) of Example 1.

[0032] Comparative Example 3 The DNA fragment mixture was prepared in the same manner as in Example 1, except that instead of adding magnesium chloride in (step 6) of Example 1, it was heat-treated at 100°C.

[0033] Comparative Example 4 The DNA fragment mixture was prepared in the same manner as in Example 1, except that 0.4% SDS was added instead of magnesium chloride in (step 6) of Example 1.

[0034] Comparative Example 5 The DNA fragment mixture was prepared in the same manner as in Example 1, except that instead of adding magnesium chloride in (step 6) of Example 1, the pH was adjusted to 4 and the mixture was treated at 85°C for 20 minutes.

[0035] Experimental Example 1: Comparison of Turbidity and Purity by the Fish Milk Impurity Removal Method The turbidity and purity of the fish milt impurity removal methods were compared for three examples: one in which isopropanol was added, Comparative Example 1 in which distilled water was added, and Comparative Example 2 in which nothing was added.

[0036] PN (1g) prepared according to each method for removing impurities from aquatic milt was hydrated with 100mL of sterile distilled water, and the turbidity was compared (Figure 1). As a result, the PN prepared using the method of adding isopropanol showed the highest transparency. On the other hand, the PN of Comparative Example 1, prepared with distilled water, and the PN of Comparative Example 2, prepared without any additives, were opaque white, with the PN of Comparative Example 2 being the most opaque. Therefore, through turbidity comparison, it was confirmed that the method of preparing the DNA fragment mixture of the present invention using isopropanol has the best effect in removing impurities from aquatic milt.

[0037] [Table 1]

[0038] As shown in Table 1, after hydrating with 100 ml of sterile distilled water, the purity of 1 g of PN prepared according to each impurity removal method was compared. The A260 / 280 value was 1.8 to 1.9, and the A260 / 230 value was 2.0 to 2.2, confirming that the purity of PN from Example 1 was the highest.

[0039] Experimental Example 2: Comparison of PN yield by the fish milt impurity removal method The PN yield was compared using the methods for removing impurities from the fish milt in the example where isopropanol was added, Comparative Example 1 where distilled water was added, and Comparative Example 2 where nothing was added.

[0040] [Table 2]

[0041] As a result, as shown in Table 2, the final PN yield after removing impurities from the fish milt was confirmed to be highest at 6% in the example prepared using the method of adding isopropanol.

[0042] Experimental Example 3: Comparison of the molecular reduction effect of nucleic acid molecule reduction methods The molecular reduction effects of the nucleic acid molecular reduction methods used in the example using magnesium chloride, Comparative Example 3 using heat treatment, Comparative Example 4 using SDS, and Comparative Example 5 with controlled pH were compared by electrophoretic analysis of the molecular weight of PN prepared by each method.

[0043] As a result, as shown in Figure 2, the most superior molecular reduction effect was observed when magnesium chloride was used, while in Comparative Examples 3 to 5, no molecular reduction effect was observed, or the effect was relatively lower compared to the Examples. Therefore, it was confirmed that the method of preparing the DNA fragment mixture of the present invention using magnesium chloride has the most superior molecular reduction effect, and thus it can be used to prepare DNA fragment mixtures with molecular weights of 1,000 to 10,000 kDa.

[0044] Experimental Example 4: Comparison of molecular reduction effects of magnesium chloride treatment time In Experimental Example 3 described above, it was confirmed that the magnesium chloride treatment method was the most effective in reducing DNA molecular weight. Therefore, the inventors further investigated the effect of magnesium chloride treatment time on reducing DNA molecular weight. For comparison, magnesium chloride was treated for 1 hour (Example 3), 2 hours (Example 2), and 3 hours 30 minutes (Example 3), and PN (HTL Co.) was used as a control.

[0045] As a result, as shown in Figures 3 and 4, the longer the magnesium chloride treatment time, the greater the effect of reducing the molecular weight of DNA. In particular, the PN from Example 1 was analyzed to have a DNA molecular weight of 325-975 kda, which was similar to that of the control PN.

[0046] Experimental Example 5: GPC Analysis Results Based on Magnesium Chloride Treatment Time Following Experimental Example 3, GPC (Gel Permeation Chromatography) was performed according to the treatment time of magnesium chloride for more accurate molecular weight comparison. Specifically, a SHODEX OHpak SB-806M HQ 300 mm column was used, and the analysis was performed under the following conditions: column temperature 40°C, flow rate 1.0 mL / min, RI detector, pressure 1.7 MPa, and 100 μl injection. The sample was dissolved in purified water to a 1% solution, diluted 25-fold with mobile phase A, filtered through a 0.45 μm filter, and then analyzed. The standards used were dextran standard 1 (2,457,000 MW), dextran standard 2 (1,150,000 MW), and dextran standard 3 (405,700 MW).

[0047] [Table 3]

[0048] As shown in Table 3 and Figures 5A to 5D, it was confirmed that increasing the magnesium chloride treatment time narrowed the range of DNA molecular weight, allowing for the preparation of a DNA fragment mixture with a consistent molecular weight, thus inducing PN purification. In addition, it was confirmed that the average molecular weight was similar to that of the control group.

[0049] Experimental Example 6: Comparison of Viscoelasticity of Example 1 and the Control The viscoelasticity of Example 1 and the control was compared. Specifically, the viscoelasticity of the samples was analyzed using a viscoelastic analyzer (MCR 92, manufacturer: Anton Paar), a spindle (measurement plate PP25 D:25 mm, manufacturer: Anton Paar), a plunger rod, and a spatula. At this time, a reference point was set using viscosity standards (Viscosity Standard 1000, Viscosity Standard 5000 - manufacturer: BROOKFIELD AMETE), and 1 ml of 1% PN (sample) dissolved in purified water was dispensed into the viscoelastic analyzer. Next, the accumulated modulus and loss modulus of each sample were measured and analyzed at a total of 16 points, with the angular frequency ω (rad / s) set to 1.

[0050] [Table 4]

[0051] As a result, as shown in Table 4 and Figure 6, the storage modulus and loss modulus of Example 1 were higher than those of the control group, confirming that Example 1 has superior viscoelasticity.

Claims

1. A method for preparing a high-purity DNA fragment mixture, comprising the following steps: (Step 1) Treat the milt with isopropanol to obtain milt from which impurities have been removed. (Step 2) The milt from which the above impurities have been removed is treated by adding it to an aqueous sodium chloride solution. (Step 3) Add an aqueous sodium lauryl sulfate solution to an aqueous sodium chloride solution containing fish milt to obtain a primary fish milt lysate solution in which the cells are broken down and nucleic acids are extracted. (Step 4) The primary milt lysate solution is precipitated with ethanol, and the precipitate is separated to obtain the secondary milt lysate solution. (Step 5) Add sodium chloride to the secondary alfonsino lysate solution and first inactivate the virus at a high temperature of 80-100°C. (Step 6) Adding magnesium chloride to the virus-inactivated alfonsino lysate solution to obtain a DNA fragment mixture with reduced nucleic acid molecular weight, and (Step 7) The DNA fragment mixture with reduced nucleic acid molecular weight is treated with ethanol to secondarily inactivate the virus, and then a precipitate is obtained. The method, including the method described above.

2. A method for preparing a high-purity DNA fragment mixture according to claim 1, wherein the isopropanol in step 1 is 3 to 6 times the weight of the fish milt.

3. Step 2 is to add a 20-50% by weight aqueous sodium chloride solution to the milt from which impurities have been removed, treat it at a temperature of 90-100°C for 1-4 hours, and then cool it, in a method for preparing a high-purity DNA fragment mixture according to claim 1.

4. Step 3 is to add an aqueous solution of sodium lauryl sulfate at a concentration of 10-20% by weight to a final concentration of 0.5-2% by weight, and to treat at 10-30°C for 20-40 minutes, a method for preparing a high-purity DNA fragment mixture according to claim 1.

5. Step 4 is a method for preparing a high-purity DNA fragment mixture according to claim 1, wherein step 4 involves adding ethanol to a final concentration of 50-80% to precipitate the primary alfonsino lysate solution, and separating the precipitate to obtain a secondary alfonsino lysate solution.

6. A method for preparing a high-purity DNA fragment mixture according to claim 1, wherein the primary virus inactivation in step 5 is carried out by adding sodium chloride to a final concentration of 0.5 to 2 M and treating at a high temperature of 80 to 100°C for 12 to 20 hours.

7. Step 6 is the method for preparing a high-purity DNA fragment mixture according to claim 1, wherein magnesium chloride is added to a final concentration of 30 to 10 mM and the mixture is treated at 80 to 100°C for 2 to 5 hours.

8. Step 7 is the method for preparing a high-purity DNA fragment mixture according to claim 1, wherein step 7 is the addition of 50-80% ethanol and the mixture is treated for 12-20 hours.

9. A method for preparing a high-purity DNA fragment mixture according to claim 1, wherein the DNA fragments have a molecular weight of 1,000 to 10,000 kDa.

10. A method for preparing a high-purity DNA fragment mixture according to claim 1, wherein the milt is salmon milt.

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