Method for manufacturing a high-purity DNA fragment mixture

Abstract

The present invention relates to a method for preparing a high-purity DNA fragment mixture using isopropyl alcohol and magnesium chloride, in which isopropyl alcohol is used to effectively remove impurities such as moisture, blood, and fat and increase the purity of the DNA fragment mixture, and magnesium chloride can be used to prepare a DNA fragment mixture having a relatively narrow molecular weight range of 1000 KDa to 10000 KDa.

Description

TECHNICAL FIELD

[0001] The present application relates to a method of manufacturing a DNA fragment mixture, and more particularly, to a method of preparing a high-purity DNA fragment mixture using isopropyl alcohol and magnesium chloride. BACKGROUND

[0002] Nucleotides are structural units that constitute nucleic acids such as DNA and RNA, and a polymer formed of 10 or more nucleotides is called a polynucleotide (PN). In addition, nucleotides are composed of one 5-carbon sugar (pentose), one phosphate group, and one base (adenine, guanine, thymine, or cytosine), and the balance of combinations of nucleotides in trout or salmon has 96.5% identity with humans. This high similarity is very important because it can be fully utilized without discarding any nucleic acids.

[0003] Meanwhile, the DNA fragment mixture is a mixture in which DNA exists in the form of a fragment with a reduced molecular weight. Since the DNA fragment mixture contains essential components of cells, its value is increasing due to its diversified uses, for example, being used for medical devices and medicines, for the purpose of treating and improving wounds, such as by being injected into wounds, musculoskeletal areas, or joints, knees, and joint cavities to reduce mechanical friction and pain, or as cosmetics, food additives, and biochemical experimental materials, for the purpose of improving wrinkles related to cell activity.

[0004] As a conventional method for preparing a DNA fragment mixture, Korean Patent Publication No. 10-390529 discloses a method of extracting nucleic acids using an ultrafiltration membrane. However, this method has the disadvantage of being difficult to extract high-purity nucleic acids because it is difficult to effectively remove impurities such as RNA, proteins, fats, and moisture. In addition, Korean Patent Publication No. 10-2019-0065676 discloses a method of 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] Accordingly, the present inventors, in researching a method for preparing a high-purity DNA fragment mixture that safely reduces the molecular weight of DNA and effectively removes impurities, confirmed that when isopropyl alcohol is used to remove impurities and magnesium chloride is used to mix DNA fragments, the molecular weight of DNA can be safely reduced, and a high yield and high purity can be obtained, thereby completing the present application. SUMMARY

[0006] An object of the present application is to provide a method for preparing a high-purity DNA fragment mixture.

[0007] Another object of the present application is to provide a high-purity DNA fragment mixture.

[0008] To achieve the above objectives, the present invention provides a method for preparing a high-purity DNA fragment mixture, the method comprising the following steps:

[0009] (Step 1) The step of treating fish essence with isopropanol to obtain fish essence with impurities removed;

[0010] (Step 2) The step of treating the fish essence with impurities removed by adding it to a sodium chloride aqueous solution;

[0011] (Step 3) Add sodium dodecyl sulfate aqueous solution to sodium chloride aqueous solution containing fish essence to obtain primary fish essence lysate in which cells are decomposed and nucleic acids are extracted.

[0012] (Step 4) Precipitate the primary fish sperm lysate with ethanol and separate the precipitate to obtain the secondary fish sperm lysate;

[0013] (Step 5) Add sodium chloride to the secondary fish sperm lysate and perform initial virus inactivation at a high temperature of 80 to 100°C;

[0014] (Step 6) The step of adding magnesium chloride to the virus-inactivated fish sperm lysate to obtain a mixture of DNA fragments with reduced nucleic acid molecular weight; and

[0015] (Step 7) The step of secondary virus inactivation by treating the mixture of DNA fragments with reduced nucleic acid molecular weight with ethanol and then obtaining the precipitate.

[0016] Furthermore, the present invention provides a high-purity DNA fragment mixture prepared by the above method.

[0017] Beneficial effects

[0018] The method for preparing a high-purity DNA fragment mixture according to the present invention has the following effects: the purity of the DNA fragment mixture is improved by effectively removing impurities such as water, blood and fat using isopropanol, and a DNA fragment mixture with a relatively narrow molecular weight range of 1000 kDa to 10000 kDa is prepared by using magnesium chloride. Attached Figure Description

[0019] Figure 1 These are photographs showing a comparison of the transparency of a mixture of DNA fragments prepared according to a pretreatment method.

[0020] Figure 2 These are photographs showing a comparison of the molecular weight reduction effects of different nucleic acid molecular weight reduction methods.

[0021] Figure 3 These are photographs showing a comparison of the molecular weight reduction effect based on magnesium chloride treatment time.

[0022] Figure 4 This is a photograph showing a comparison of the molecular weights of Example 1 and the control group (PN, HTL).

[0023] Figure 5a This is a graph showing a comparison of GPC measurement results with those of the control group.

[0024] Figure 5b This is a graph showing a comparison of the GPC measurement results of Example 3.

[0025] Figure 5c This is a graph showing a comparison of the GPC measurement results of Example 2.

[0026] Figure 5d This is a graph showing a comparison of the GPC measurement results of Example 1.

[0027] Figure 6 This is a graph showing the viscoelasticity comparison between Example 1 and the control group. Detailed Implementation

[0028] The present invention is described in detail below.

[0029] In one aspect of the invention, a method for preparing a mixture of high-purity DNA fragments is provided, the method comprising the following steps:

[0030] (Step 1) The step of treating fish essence with isopropanol to obtain fish essence with impurities removed;

[0031] (Step 2) The step of treating the fish essence with impurities removed by adding it to a sodium chloride aqueous solution;

[0032] (Step 3) Add sodium dodecyl sulfate aqueous solution to sodium chloride aqueous solution containing fish essence to obtain primary fish essence lysate in which cells are decomposed and nucleic acids are extracted.

[0033] (Step 4) Precipitate the primary fish sperm lysate with ethanol and separate the precipitate to obtain the secondary fish sperm lysate;

[0034] (Step 5) Add sodium chloride to the secondary fish sperm lysate and perform initial virus inactivation at a high temperature of 80 to 100°C;

[0035] (Step 6) The step of adding magnesium chloride to the virus-inactivated fish sperm lysate to obtain a mixture of DNA fragments with reduced nucleic acid molecular weight; and

[0036] (Step 7) The step of secondary virus inactivation by treating the mixture of DNA fragments with reduced nucleic acid molecular weight with ethanol and then obtaining the precipitate.

[0037] In step 1 above, isopropanol is added to fish essence, the mixture of fish essence and isopropanol is crushed, the mixture is left to stand for 5 to 30 minutes, and then the crushed fish essence and isopropanol are separated to complete the removal of impurities.

[0038] 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 fish essence.

[0039] The fish essence can be from trout or salmon, preferably salmon.

[0040] Steps 2 and 3 above are steps for cell decomposition and nucleic acid extraction. In step 2, cells are decomposed at high temperature using a heat treatment method, and nucleic acids are extracted using sodium chloride. Furthermore, in step 3, cells not decomposed in step 2 are decomposed using sodium dodecyl sulfate, and impurities including fats and proteins are further removed.

[0041] More specifically, in step 2 above, a sodium chloride aqueous solution with a concentration of 20 wt% to 50 wt% is added to the fish essence to which impurities have been removed, and the mixture is treated at a temperature of 90°C to 100°C for 1 to 4 hours, and then cooled to a temperature of 60°C to 65°C. Preferably, a sodium chloride aqueous solution with a concentration of 25 wt% to 35 wt% is added, followed by treatment at a temperature of 95°C to 100°C for 1 to 2 hours.

[0042] In step 3 above, a 10 wt% to 20 wt% aqueous solution of sodium dodecyl sulfate is added to an aqueous solution of sodium chloride containing fish essence to bring the final concentration to 0.5 wt% to 2 wt%, and the mixture is treated at 10°C to 30°C for 20 to 40 minutes. Preferably, the sodium dodecyl sulfate aqueous solution is added to bring the final concentration to 1 wt% to 1.5 wt%.

[0043] In step 4 above, the primary fish sperm lysate is precipitated by adding ethanol to a final concentration of 50% to 80%, and the precipitate is separated to obtain the secondary fish sperm lysate.

[0044] More specifically, the primary fish protease lysate was filtered through a filter with a pore size of 50 μm to 10 μm. Ethanol was added to the filtrate to a final concentration of 50% to 80%, and the solution was allowed to stand for 20 minutes to separate the precipitate. The precipitate was then dissolved in distilled water to obtain the secondary fish protease lysate.

[0045] The initial virus inactivation in step 5 involves adding sodium chloride to a final concentration of 0.5M to 2M and treating at a high temperature of 80°C to 100°C for 12 to 20 hours. Preferably, sodium chloride is added to a final concentration of 0.7M to 1.5M, and the treatment can be carried out at a high temperature of 80°C to 90°C.

[0046] Step 6 involves adding magnesium chloride to achieve a final concentration of 10 mM to 30 mM and treating at 80°C to 100°C for 2 to 5 hours. Preferably, magnesium chloride is added to achieve a final concentration of 15 mM to 35 mM, and the treatment is carried out at a high temperature of 80°C to 90°C.

[0047] In step 6 above, magnesium chloride breaks down nucleic acids and reduces the molecular weight of DNA fragments.

[0048] Step 7 involves adding 50% to 80% ethanol and treating for 12 to 20 hours.

[0049] Specifically, in this step, ethanol is added to a final concentration of 50% to 80%, and the mixture is allowed to stand for 10 to 30 minutes to separate the precipitate. Then, 50% to 80% ethanol is added to the precipitate and the mixture is treated for 12 to 20 hours. Preferably, 60% to 75% ethanol is added and the mixture is treated for 15 to 16 hours.

[0050] In another aspect of the invention, the invention provides a high-purity DNA fragment mixture prepared by the above method.

[0051] The DNA fragment is characterized by a molecular weight of 1000 kDa to 10000 kDa.

[0052] In this invention, "DNA fragment mixture" refers to PDRN (polydeoxyribonucleotide).

[0053] In this invention, "impurities" include blood, water, and proteins, fats, endotoxins, etc., washed out due to the decomposition of fish sperm cells.

[0054] In this invention, "processing" refers to soaking or stirring.

[0055] The present invention will be described in detail below through the following embodiments and experimental examples.

[0056] However, the following embodiments and experimental examples are only used to illustrate the present invention, and the content of the present invention is not limited thereto.

[0057] Example 1: Preparation of high-purity DNA fragment mixture 1

[0058] Prepare a high-purity DNA fragment mixture by following these steps.

[0059] (Step 1) After thawing 200g of frozen salmon essence, remove blood vessels, blood, eggs, and other internal organs. Add isopropyl alcohol at a ratio of 5 times the weight of the salmon essence to the above salmon essence, grind with a stirrer, and let stand for 10 minutes. Then, separate the salmon essence powder and isopropyl alcohol by centrifugation and filtration.

[0060] (Step 2) Add sterile distilled water to the pulverized material from Step 1 and stir. Then add 30% sodium chloride and stir slowly at 95°C for 1 hour. Then cool the reaction mixture to 60-65°C.

[0061] (Step 3) Add sodium dodecyl sulfate to the sodium chloride aqueous solution containing fish essence until the final concentration is 1%, and stir slowly for 30 minutes.

[0062] (Step 4) After filtering the lysis solution from Step 3 through a filter with a pore size of 50 to 10 micrometers, add ethanol to the filtrate to a final concentration of 50% to 80% and let it stand for 20 minutes. After standing, wash the obtained precipitate with 50% to 80% ethanol while stirring slowly. After washing, dehydrate the precipitate in 95% ethanol and dry it using a hot air dryer (55°C) or a vacuum dryer (45°C, -0.09 to -0.1 MPa). Then, completely dissolve the dried product in sterile distilled water to a final concentration of 1% to 3%.

[0063] (Step 5) Filter the lysis solution from step 4 through a filter with a diameter of 1 to 0.5 micrometers, add sodium chloride to it until the final concentration is 1M, and then stir slowly at 85°C for 16 hours.

[0064] (Step 6) Add magnesium chloride to the lysis solution from Step 5 to a final concentration of 20 mM and stir slowly at 85°C for 3 hours and 30 minutes.

[0065] (Step 7) The lysis buffer from Step 6 above is aseptically filtered through a filter with a pore size of 0.22 micrometers. Ethanol is added to the filtrate to a final concentration of 70%, and the mixture is allowed to stand for 20 minutes to obtain a precipitate. The precipitate is washed by adding 70% ethanol and stirring slowly for 16 hours.

[0066] (Step 8) Add 95% ethanol to the precipitate washed in Step 7 above, stir the mixture slowly for 10 minutes to dehydrate, and then dry it with a hot air dryer (55°C) or a vacuum dryer (45°C, -0.09 to -0.1 MPa) to prepare a high-purity DNA fragment mixture with a molecular weight of 1000 to 10000 kDa.

[0067] Example 2: Preparation of high-purity DNA fragment mixture 2

[0068] 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 performed at 85°C for 2 hours.

[0069] Example 3: Preparation of high-purity DNA fragment mixture 3

[0070] 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 performed at 85°C for 1 hour.

[0071] Comparative Example 1

[0072] 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.

[0073] Comparative Example 2

[0074] 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.

[0075] Comparative Example 3

[0076] The DNA fragment mixture was prepared in the same manner as in Example 1, except that step 6 of Example 1 was performed by heat treatment at 100°C without the addition of magnesium chloride.

[0077] Comparative Example 4

[0078] 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.

[0079] Comparative Example 5

[0080] The DNA fragment mixture was prepared in the same manner as in Example 1, except that magnesium chloride was not added in step 6 of Example 1, and the pH was adjusted to 4 and treated at 85°C for 20 minutes.

[0081] Experimental Example 1: Comparison of turbidity and purity according to the protamine impurity removal method

[0082] The turbidity and purity of the fish essence impurity removal methods according to the example with the addition of isopropanol, Comparative Example 1 with the addition of distilled water, and Comparative Example 2 without the addition of any substance were compared.

[0083] PN (1g) prepared based on each fish sperm impurity removal method was hydrated in 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 adding any substance were both opaque white, with the PN of Comparative Example 2 being the least transparent. Therefore, by comparing the turbidity, it was confirmed that the method of preparing DNA fragment mixtures using isopropanol of the present invention has the best effect on removing fish sperm impurities.

[0084] [Table 1]

[0085] Absorbance Example 1 Comparative Example 1 Comparative Example 2 600 nm 0.0034 0.0600 0.8300 280 nm 0.5704 0.6704 0.3704 260 nm 1.0340 1.1340 0.6034 230 nm 0.4825 0.4925 0.2925 260 / 280 1.81 1.69 1.63 260 / 230 2.14 2.30 2.06

[0086] As shown in Table 1, the results of comparing the purity of 1g of PN prepared according to each impurity removal method after hydration in 100ml of sterile distilled water confirmed that the PN of Example 1 had the best purity, with an A260 / 280 value of 1.8 to 1.9 and an A260 / 230 value of 2.0 to 2.2.

[0087] Experimental Example 2: Comparison of PN yield according to the protamine impurity removal method

[0088] The PN yield of the fish sperm impurity removal method was compared with that of the example with the addition of isopropanol, Comparative Example 1 with the addition of distilled water and Comparative Example 2 without the addition of any substance.

[0089] [Table 2]

[0090] Final PN yield Example 6％(12g) Comparative Example 1 4％(8g) Comparative Example 2 4％(8g)

[0091] The results, as shown in Table 2, demonstrate the final PN yield according to the fish sperm impurity removal method, confirming that the PN yield of the example prepared using the method of adding isopropanol was the highest, at 6%.

[0092] Experiment Example 3: Comparison of molecular weight reduction effects based on nucleic acid molecular weight reduction methods

[0093] The molecular weight reduction effects of nucleic acid molecular weight reduction methods according to the examples using magnesium chloride, comparative example 3 using heat treatment, comparative example 4 using SDS, and comparative example 5 controlling pH were compared, and the molecular weight of PN prepared by each method was analyzed by electrophoresis.

[0094] As a result, Figure 2 As shown, the best molecular weight reduction effect was observed when magnesium chloride was used, while in Comparative Examples 3 to 5, no molecular weight reduction effect was observed, or the observed effect was relatively low compared to the examples. Therefore, it is confirmed that the method of the present invention for preparing DNA fragment mixtures using magnesium chloride has the best molecular weight reduction effect and can therefore be used to produce DNA fragment mixtures with molecular weights of 1000 to 10000 kDa.

[0095] Experiment Example 4: Comparison of molecular weight reduction effects based on magnesium chloride treatment time

[0096] In Experiment 3 above, it was confirmed that the magnesium chloride treatment method had the best DNA molecular weight reduction effect. Therefore, the inventors further confirmed the DNA molecular weight reduction effect based on the magnesium chloride treatment time. For comparison, magnesium chloride was used for 1 hour (Example 3), 2 hours (Example 2), and 3 hours and 30 minutes (Example 3), respectively, with PN (HTL Corporation) used as a control.

[0097] As a result, Figure 3 and Figure 4 As shown, the longer the magnesium chloride treatment time, the better the reduction in DNA molecular weight. In particular, the DNA molecular weight of PN in Example 1 was analyzed and found to be 325 to 975 kDa, similar to that of the control PN.

[0098] Experimental Example 5: GPC analysis results according to magnesium chloride treatment time

[0099] According to Experimental Example 3, to more accurately compare molecular weights, GPC (gel permeation chromatography) based on magnesium chloride treatment time was performed. Specifically, the analysis was conducted 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 injection volume 100 μL. The sample was dissolved in purified water to 1%, diluted 25-fold in 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).

[0100] [Table 3]

[0101] Minimum molecular weight Maximum molecular weight Average molecular weight Control 947 4312 2291 Example 3 253 6563 3959 Example 2 190 4921 3116 Example 1 766 4923 2014

[0102] As shown in Table 3 and Figures 5a to 5d As shown, this confirms that the molecular weight range of DNA decreases with increasing magnesium chloride treatment time, resulting in a mixture of DNA fragments with constant molecular weight, thereby inducing PN purification. Furthermore, it confirms that the PN fragments have a similar average molecular weight to the control.

[0103] Experimental Example 6: Comparison of viscoelasticity of Example 1 and the control

[0104] The viscoelasticity of Example 1 was compared with that of the control. Specifically, the viscoelasticity of the samples was analyzed using a viscoelastic apparatus (MCR 92, manufacturer: Anton Paar), a rotor (measuring plate PP25 D:25mm, manufacturer: Anton Paar), a plunger rod, and a scraper. Reference points were established using viscosity standards (Viscosity Standard 1000, Viscosity Standard 5000 - manufacturer: BROOKFIELD AMETE), and 1 ml of 1% PN (sample) dissolved in pure water was dispensed into the viscoelastic apparatus. The storage modulus and loss modulus of each sample were then measured at a total of 16 points, analyzed at an angular frequency ω (rad / s) of 1.

[0105] [Table 4]

[0106]

[0107] The results are shown in Table 4 and Figure 6 As shown, since the storage modulus and loss modulus of Example 1 are higher than those of the control, it is confirmed that Example 1 has better viscoelasticity.

Claims

1. A method for preparing a mixture of high-purity DNA fragments, the method comprising the following steps: (Step 1) Treat the fish essence with isopropanol in an amount of 3 to 6 times the weight of the fish essence for 5 to 30 minutes to obtain fish essence with impurities removed. (Step 2) Add a sodium chloride aqueous solution with a concentration of 20wt% to 50wt% to the fish essence with impurities removed above, and treat it at 90°C to 100°C for 1 hour to 4 hours, and then cool it. (Step 3) Add a sodium dodecyl sulfate aqueous solution with a concentration of 10 wt% to 20 wt% until the final concentration is 0.5 wt% to 2 wt%, and treat at 10°C to 30°C for 20 to 40 minutes to obtain a primary fish sperm lysate in which cells are decomposed and nucleic acids are extracted. (Step 4) The primary fish sperm lysate is precipitated by adding ethanol until the final concentration is 50% v / v to 80% v / v and treating for 20 minutes, and the precipitate is separated to obtain the secondary fish sperm lysate. (Step 5) Add sodium chloride to the secondary fish sperm lysate until the final concentration is 0.5M to 2M and treat at 80°C to 100°C for 12 to 20 hours to inactivate the virus for the first time. (Step 6) Add magnesium chloride to the virus-inactivated fish sperm lysate until the final concentration is 10 mM to 30 mM, and treat at 80°C to 100°C for 2 to 5 hours to obtain a mixture of DNA fragments with reduced nucleic acid molecular weight; and (Step 7) Add ethanol until the final concentration is 50% v / v to 80% v / v, let stand for 10 to 30 minutes to separate the precipitate, and treat the precipitate with 50% v / v to 80% v / v ethanol for 12 to 20 hours to perform a secondary virus inactivation step.

2. The method for preparing a high-purity DNA fragment mixture according to claim 1, wherein the molecular weight of the DNA fragment is from 1000 kDa to 10000 kDa.

3. The method for preparing a high-purity DNA fragment mixture according to claim 1, wherein the fish essence is salmon fish essence.

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