Method for preparing a highly pure 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 DNA fragment mixture, specifically to a method for preparing a highly pure DNA fragment mixture using isopropanol and magnesium chloride. [Background technology]

[0002] 2. Description of Related Technology Nucleotides are the structural units that make up nucleic acids such as DNA and RNA, and a polymer consisting of 10 or more nucleotides is called a polynucleotide (PN). Additionally, nucleotides are composed of a combination of one 5-carbon sugar (pentose), one phosphate, and one base (adenine, guanine, thymine, or cytosine). The balance of the nucleotide combinations that make up trout and salmon nucleotides is 96.5% identical to that of humans. This high similarity is important because it allows the entire nucleic acid to be used without waste.

[0003] On the other hand, DNA fragment mixtures are mixtures in which DNA exists in the form of fragments with reduced molecular weight. Because DNA fragment mixtures contain essential components of cells, they are becoming increasingly valuable for a variety of applications, such as medical devices and pharmaceuticals for wound treatment and improvement, such as injecting them into wounds, musculoskeletal areas, or joints, knees, and articular cavities to relieve mechanical friction and pain, or as cosmetics, food additives, and biochemical experimental materials for improving wrinkles related to cellular activity.

[0004] Korean Patent Publication No. 10-390529 discloses a method for extracting nucleic acids using an ultrafiltration membrane as a conventional method for preparing a DNA fragment mixture. However, this method has the disadvantage of being unable to effectively remove impurities such as RNA, protein, fat, and water, making it difficult to extract highly pure 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 is difficult and dangerous because it uses strong acids or strong bases.

[0005] Therefore, the present inventors investigated a method for preparing a high-purity DNA fragment mixture that safely reduces the molecular weight of DNA and effectively removes impurities, and confirmed that using isopropanol to remove impurities and magnesium chloride to mix the DNA fragments allows the molecular weight of DNA to be safely reduced with high yield and high purity, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0006] Summary of the Invention An object of the present invention is to provide a method for preparing a highly pure DNA fragment mixture. Another object of the present invention is to provide a highly pure mixture of DNA fragments.

[0007] To achieve the above object, the present invention provides a method for preparing a highly pure DNA fragment mixture, comprising the following steps: (Step 1) treating the milt with isopropanol to obtain milt from which impurities have been removed; (Step 2) treating the milt from which the impurities have been removed by adding it to an aqueous sodium chloride solution; (Step 3) adding a sodium lauryl sulfate aqueous solution to the sodium chloride aqueous solution containing the milt to obtain a primary milt lysate solution in which the cells are lysed and nucleic acids are extracted; (Step 4) Precipitating the primary milt lysate solution with ethanol and separating the precipitate to obtain a secondary milt lysate solution; (Step 5) Add sodium chloride to the secondary milt lysate solution and inactivate the virus at a high temperature of 80-100°C. (Step 6) adding magnesium chloride to the virus-inactivated milt lysate solution to obtain a DNA fragment mixture with reduced nucleic acid molecular weight; and (Step 7) A step of secondary inactivating viruses by treating the DNA fragment mixture with ethanol to reduce the nucleic acid molecular weight of the DNA fragments, and then obtaining a precipitate.

[0008] Additionally, the present invention provides a highly pure DNA fragment mixture prepared by the above method. [Effects of the Invention]

[0009] beneficial effects The method for preparing a highly pure DNA fragment mixture according to the present invention has the following advantages: by using isopropanol, impurities such as water, blood, and fat are effectively removed, thereby increasing the purity of the DNA fragment mixture; and by using magnesium chloride, a DNA fragment mixture having a relatively narrow molecular weight range of 1,000 kDa to 10,000 kDa is prepared. [Brief explanation of the drawings]

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

[0011] Description of the Preferred Embodiment The present invention will be described in detail below. In one aspect, the present invention provides a method for preparing a highly pure DNA fragment mixture, comprising the steps of: (Step 1) treating the milt with isopropanol to obtain milt from which impurities have been removed; (Step 2) treating the milt from which the impurities have been removed by adding it to an aqueous sodium chloride solution; (Step 3) adding a sodium lauryl sulfate aqueous solution to the sodium chloride aqueous solution containing the milt to obtain a primary milt lysate solution in which the cells are lysed and nucleic acids are extracted; (Step 4) Precipitating the primary milt lysate solution with ethanol and separating the precipitate to obtain a secondary milt lysate solution; (Step 5) Add sodium chloride to the secondary milt lysate solution and inactivate the virus at a high temperature of 80-100°C. (Step 6) adding magnesium chloride to the virus-inactivated milt lysate solution to obtain a DNA fragment mixture with reduced nucleic acid molecular weight; and (Step 7) A step of secondary inactivating viruses by treating the DNA fragment mixture with ethanol to reduce the nucleic acid molecular weight of the DNA fragments, and then obtaining a precipitate.

[0012] In step 1 above, impurities are removed by adding isopropanol to the milt, grinding the mixture of milt and isopropanol, leaving the mixture for 5 to 30 minutes, and then separating the milt grind and the isopropanol. The isopropanol can be added in an amount of 3 to 6 times, preferably 4 to 5 times, and more preferably 5 times the weight of the milt. The milt may be trout milt or salmon milt, and is preferably salmon milt.

[0013] Steps 2 and 3 above are steps for cell decomposition and nucleic acid extraction. In step 2, cells are decomposed at high temperature via heat treatment, and nucleic acids are extracted using sodium chloride. In step 3, sodium lauryl sulfate is used to decompose cells that were not decomposed in step 2 and to remove impurities such as fat and protein.

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

[0015] In the above step 3, 10 to 20% by weight of an aqueous sodium lauryl sulfate solution is added to the aqueous sodium chloride solution containing the milt to give a final concentration of 0.5 to 2% by weight, and the mixture is 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 is 1 to 1.5% by weight.

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

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

[0018] The first viral inactivation step in step 5 involves 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. Preferably, sodium chloride is added to a final concentration of 0.7 to 1.5 M and treatment is performed at a high temperature of 80 to 90°C.

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

[0020] Step 7 is a step in which 50 to 80% ethanol is added and treatment is carried out for 12 to 20 hours. Specifically, in this step, ethanol is added to a final concentration of 50-80%, left to stand for 10-30 minutes, the precipitate is separated, 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, the present invention provides a highly pure 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, the term "DNA fragment mixture" refers to PN (polynucleotide) or PDRN (polydeoxyribonucleotide), and preferably refers to PN.

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

[0023] The present invention will now be described in detail with reference to the following examples and experimental examples. However, the following examples and experimental examples are merely for the purpose of illustrating the present invention, and the contents of the present invention are not limited thereto.

[0024] Example 1: Preparation of Highly Pure DNA Fragment Mixture 1 A highly pure DNA fragment mixture was prepared according to the following procedure. (Step 1) 200 g of frozen salmon milt was thawed, and then internal organs such as blood vessels, blood, and eggs were removed. An amount of isopropanol equivalent to five times the weight of the milt was added to the milt, which was then pulverized in a mixer and allowed to stand for 10 minutes. The milt powder and isopropanol were then separated by centrifugation and filtration. (Step 2) Sterile distilled water was added to the pulverized material from Step 1 and stirred, and then 30% sodium chloride was added and stirred slowly for 1 hour at 95°C. The reaction mixture was then cooled to 60-65°C.

[0025] (Step 3) Sodium lauryl sulfate was added to the aqueous sodium chloride solution containing the milt to a final concentration of 1%, and the mixture was slowly stirred for 30 minutes. (Step 4) After filtering the lysate solution from Step 3 above through a 50-10 μm pore size filter, ethanol was added to the filtrate to a final concentration of 50-80% and allowed to stand for 20 minutes. After standing, the resulting precipitate was washed by adding 50-80% ethanol and slowly stirring. 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 above was filtered through a 1-0.5 μm filter, and sodium chloride was added to the solution to a final concentration of 1 M, followed by gentle stirring at 85°C for 16 hours. (Step 6) Magnesium chloride was added to the lysate solution from Step 5 above to a final concentration of 20 mM, and the mixture was stirred slowly at 85°C for 3 hours and 30 minutes.

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

[0028] Example 2: Preparation of a highly pure DNA fragment mixture 2 A highly pure 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 highly pure DNA fragment mixture 3 A highly pure 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 heat treatment at 100°C was carried out instead of adding magnesium chloride in (step 6) of Example 1.

[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 using the milt impurity removal method The turbidity and purity of the milt impurity removal methods were compared among the Example 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 (1 g) prepared according to each milt impurity removal method was hydrated with 100 mL of sterile distilled water, and the turbidity was compared (Figure 1). As a result, the PN of the Example, prepared using the method of adding isopropanol, was the most transparent. On the other hand, the PN of Comparative Example 1, prepared by adding distilled water, and the PN of Comparative Example 2, prepared without adding anything, were opaque white, with the PN of Comparative Example 2 being the most opaque. Therefore, through the turbidity comparison, it was confirmed that the method of preparing a DNA fragment mixture of the present invention using isopropanol has the most excellent effect on removing milt impurities.

[0037] [Table 1]

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

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

[0040] [Table 2]

[0041] As a result, as shown in Table 2, the final PN yields according to the method for removing milt impurities were confirmed to be the highest at 6% in the example prepared using the method of adding isopropanol.

[0042] Experimental Example 3: Comparison of molecule reduction effects by nucleic acid molecule reduction method The molecular weight reduction effects of the nucleic acid molecule reduction methods of Example 1 using magnesium chloride, Comparative Example 3 using heat treatment, Comparative Example 4 using SDS, and Comparative Example 5 controlling the pH were compared by electrophoretic analysis of the molecular weight of the PN prepared by each method.

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

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

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

[0046] Experimental Example 5: GPC analysis results depending on magnesium chloride treatment time Following Experimental Example 3, for more accurate molecular weight comparisons, GPC (gel permeation chromatography) was performed depending on the magnesium chloride treatment time. Specifically, analysis was performed using a SHODEX OHpak SB-806M HQ 300 mm column 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% concentration, diluted 25-fold with mobile phase A, and filtered through a 0.45 μm filter before analysis. 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-5D, as the magnesium chloride treatment time increased, the range of DNA molecular weights narrowed, enabling the preparation of a DNA fragment mixture with a consistent molecular weight, confirming the induction of PN purification. In addition, it was confirmed that the average molecular weights were similar compared to the control group.

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

[0050] [Table 4]

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

Claims

1. 1. A method for preparing a highly pure DNA fragment mixture, comprising the steps of: (Step 1) treating the milt with isopropanol to obtain milt from which impurities have been removed; (Step 2) treating the milt from which the impurities have been removed by adding it to an aqueous sodium chloride solution; (Step 3) adding a sodium lauryl sulfate aqueous solution to the sodium chloride aqueous solution containing the milt to obtain a primary milt lysate solution in which the cells are lysed and nucleic acids are extracted; (Step 4) precipitating the primary milt lysate solution with ethanol and separating the precipitate to obtain a secondary milt lysate solution; (Step 5) adding sodium chloride to the secondary milt lysate solution and first inactivating the virus at a high temperature of 80-100°C; (Step 6) adding magnesium chloride to the virus-inactivated milt lysate solution to obtain a DNA fragment mixture with reduced nucleic acid molecular weight; and (Step 7) Secondary virus inactivation is performed by treating the DNA fragment mixture with ethanol to reduce the nucleic acid molecular weight of the DNA fragments, followed by obtaining a precipitate. The method comprising:

2. 2. The method for preparing a highly pure DNA fragment mixture according to claim 1, wherein the amount of isopropanol in step 1 is 3 to 6 times the weight of the milt.

3. 2. The method for preparing a highly pure DNA fragment mixture according to claim 1, wherein step 2 comprises adding an aqueous sodium chloride solution having a concentration of 20 to 50% by weight to the milt from which impurities have been removed, treating the mixture at a temperature of 90 to 100°C for 1 to 4 hours, and then cooling the mixture.

4. 2. The method for preparing a highly pure DNA fragment mixture according to claim 1, wherein step 3 comprises adding an aqueous solution of sodium lauryl sulfate having a concentration of 10 to 20% by weight to a final concentration of 0.5 to 2% by weight, and treating at 10 to 30°C for 20 to 40 minutes.

5. 2. The method for preparing a high-purity DNA fragment mixture according to claim 1, wherein step 4 comprises precipitating the primary milt lysate solution by adding ethanol to a final concentration of 50 to 80%, and separating the precipitate to obtain a secondary milt lysate solution.

6. 2. The method for preparing a highly pure 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 the mixture at a high temperature of 80 to 100°C for 12 to 20 hours.

7. 2. The method for preparing a highly pure DNA fragment mixture according to claim 1, wherein step 6 comprises adding magnesium chloride to a final concentration of 30 to 10 mM and treating at 80 to 100° C. for 2 to 5 hours.

8. 2. The method for preparing a highly pure DNA fragment mixture according to claim 1, wherein step 7 comprises adding 50 to 80% ethanol and treating for 12 to 20 hours.

9. 2. The 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. 2. The method for preparing a highly pure DNA fragment mixture according to claim 1, wherein the milt is salmon milt.

11. A highly pure DNA fragment mixture prepared by the method of claim 1.

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