Method for producing high-purity DNA fragment mixture

Abstract

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

Description

Technical Field

[0001] The present invention relates to a method for preparing a DNA fragment mixture, and in particular to a method for preparing a high-purity DNA fragment mixture by using isopropanol and magnesium chloride. Background Art

[0002] Nucleotides are the structural units that make up nucleic acids such as DNA and RNA, and polymers formed by 10 or more nucleotides are called polynucleotides (PN). In addition, nucleotides are composed of the following combinations: a 5-carbon sugar (pentose), a phosphate group (phosphate) and a base (adenine, guanine, thymine or cytosine), and the balance of combinations that make up nucleotides in trout or salmon is 96.5% identical to that in 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 fragments with reduced molecular weights. Since the DNA fragment mixture contains essential components of cells, its value is increasing due to its diverse uses, such as being used in medical devices and drugs for the purpose of treating and improving wounds, such as by injection 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 associated with cell activity.

[0004] As a conventional method for preparing a mixture of DNA fragments, 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 difficult to extract high-purity nucleic acids because it is difficult to effectively remove impurities such as RNA, protein, fat, and moisture. In addition, Korean Patent Publication No. 10-2019-0065676 discloses a method for reducing the molecular weight of DNA by adjusting pH, but this method has the disadvantage of being difficult and dangerous because it uses strong acids and strong bases.

[0005] Therefore, the present inventors, while studying a method for preparing a high-purity DNA fragment mixture in which the DNA molecular weight is safely reduced and impurities are effectively removed, confirmed that when isopropanol is used to remove impurities and magnesium chloride is used to mix the DNA fragments, the DNA molecular weight can be safely reduced and a high yield and high purity can be obtained, thereby completing the present invention. Summary of the invention

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

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

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

[0009] (Step 1) treating fish sperm with isopropyl alcohol to obtain fish sperm with impurities removed;

[0010] (Step 2) treating the fish essence after the impurities are removed by adding it to a sodium chloride aqueous solution;

[0011] (Step 3) adding an aqueous sodium dodecyl sulfate solution to an aqueous sodium chloride solution containing fish sperm to obtain a primary fish sperm lysate in which cells are decomposed and nucleic acids are extracted;

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

[0013] (Step 5) adding sodium chloride to the secondary fish sperm lysate and performing a first virus inactivation step at a high temperature of 80 to 100° C.;

[0014] (Step 6) 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) A step of secondary virus inactivation by treating the DNA fragment mixture whose nucleic acid molecular weight has been reduced with ethanol and then obtaining a precipitate.

[0017] Furthermore, the present invention provides a high-purity DNA fragment mixture prepared by the above method. 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 moisture, blood and fat by using isopropanol, and a DNA fragment mixture with a relatively narrow molecular weight range of 1000KDa to 10000KDa is prepared by using magnesium chloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 are photographs showing a comparison of the transparency of DNA fragment mixtures prepared according to the pretreatment methods.

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

[0021] Figure 3 It is a photograph which shows the comparison of the molecular weight reduction effect according to the magnesium chloride treatment time.

[0022] Figure 4 It is a photograph showing the molecular weight comparison between Example 1 and the control group (PN, HTL Corporation).

[0023] Figure 5a It is a graph showing the comparison of the GPC measurement results 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 It is a graph showing the comparison of viscoelasticity between Example 1 and the control group. DETAILED DESCRIPTION

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

[0030] In one aspect of the present invention, the present invention provides a method for preparing a high-purity DNA fragment mixture, the method comprising the following steps:

[0031] (Step 1) treating fish sperm with isopropyl alcohol to obtain fish sperm with impurities removed;

[0032] (Step 2) treating the fish essence after the impurities are removed by adding it to a sodium chloride aqueous solution;

[0033] (Step 3) adding an aqueous sodium dodecyl sulfate solution to an aqueous sodium chloride solution containing fish sperm to obtain a primary fish sperm lysate in which cells are decomposed and nucleic acids are extracted;

[0034] (Step 4) Precipitating the primary fish sperm lysate with ethanol and separating the precipitate to obtain a secondary fish sperm lysate;

[0035] (Step 5) adding sodium chloride to the secondary fish sperm lysate and performing a first virus inactivation step at a high temperature of 80 to 100° C.;

[0036] (Step 6) adding magnesium chloride to the virus-inactivated fish sperm lysate to obtain a mixture of DNA fragments with reduced nucleic acid molecular weight; and

[0037] (Step 7) A step of secondary virus inactivation by treating the DNA fragment mixture whose nucleic acid molecular weight has been reduced with ethanol and then obtaining a precipitate.

[0039] In the above step 1, impurity removal is completed by adding isopropyl alcohol to fish essence, crushing the fish essence and isopropyl alcohol mixture, letting the mixture stand for 5 to 30 minutes, and then separating the fish essence crushed material and isopropyl alcohol.

[0040] The isopropyl alcohol may be added in an amount of 3 to 6 times, preferably 4 to 5 times, more preferably 5 times the weight of the fish essence.

[0041] The fish sperm can be the fish sperm of trout or salmon, preferably the fish sperm of salmon.

[0043] The above steps 2 and 3 are steps for performing cell decomposition and nucleic acid extraction. In the above step 2, cells are decomposed at high temperature by a heat treatment method, and nucleic acids are extracted using sodium chloride. In addition, in step 3, cells that were not decomposed in step 2 are decomposed using sodium dodecyl sulfate, and impurities including fat and protein are further removed.

[0044] More specifically, in the above step 2, a sodium chloride aqueous solution having a concentration of 20 wt % to 50 wt % is added to the fish essence from which impurities have been removed, 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 having 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.

[0045] In the above step 3, 10 wt% to 20 wt% of sodium dodecyl sulfate aqueous solution is added to the sodium chloride aqueous solution containing fish essence to a final concentration of 0.5 wt% to 2 wt%, and the mixture is treated at 10° C. to 30° C. for 20 minutes to 40 minutes. Preferably, the sodium dodecyl sulfate aqueous solution is added to a final concentration of 1 wt% to 1.5 wt%.

[0047] In the above step 4, 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 a secondary fish sperm lysate.

[0048] More specifically, the primary fish sperm lysate is filtered through a filter having a pore size of 50 μm to 10 μm, ethanol is added to the filtrate to a final concentration of 50% to 80%, and the solution is allowed to stand for 20 minutes to separate the precipitate. Then, the precipitate is dissolved in distilled water to obtain the secondary fish sperm lysate.

[0050] The first virus inactivation in step 5 is to add sodium chloride to a final concentration of 0.5M to 2M, and treat at a high temperature of 80°C to 100°C for 12 hours to 20 hours. Preferably, sodium chloride is added to a final concentration of 0.7M to 1.5M, and the treatment can be performed at a high temperature of 80°C to 90°C.

[0052] Step 6 is a step of adding magnesium chloride to 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 a final concentration of 15 mM to 35 mM and treated at a high temperature of 80° C. to 90° C.

[0053] In the above step 6, magnesium chloride decomposes nucleic acid and reduces the molecular weight of DNA fragments.

[0055] Step 7 is a step of adding 50% to 80% ethanol and treating for 12 hours to 20 hours.

[0056] 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, and then 50 to 80% ethanol is added to the precipitate and treated for 12 to 20 hours. Preferably, 60 to 75% ethanol is added and treated for 15 to 16 hours.

[0057] In another aspect of the present invention, the present invention provides a high-purity DNA fragment mixture prepared by the above method.

[0058] The DNA fragment is characterized by a molecular weight of 1000 KDa to 10000 KDa.

[0060] In the present invention, "DNA fragment mixture" refers to PN (polynucleotide) or PDRN (polydeoxyribonucleotide), preferably PN.

[0061] In the present invention, "impurities" include blood, water, and proteins, fats, endotoxins, etc. eluted due to the decomposition of fish sperm cells.

[0062] In the present invention, "treating" means soaking or stirring.

[0064] Hereinafter, the present invention will be described in detail through the following Examples and Experimental Examples.

[0065] However, the following embodiments and experimental examples are only used to illustrate the present invention, and the contents of the present invention are not limited thereto.

[0067] Example 1: Preparation of High-Purity DNA Fragment Mixture 1

[0068] Follow the steps below to prepare a high-purity DNA fragment mixture.

[0069] (Step 1) After thawing 200 g of frozen salmon sperm, remove blood vessels, blood, eggs and other internal organs. Add 5 times the weight of isopropyl alcohol to the sperm, grind with a stirrer, and let stand for 10 minutes. Then, separate the sperm powder and isopropyl alcohol by centrifugation and filtration.

[0070] (Step 2) Sterile distilled water was added to the pulverized product of the above step 1 and stirred, and then 30% sodium chloride was added and slowly stirred at 95°C for 1 hour. The reaction mixture was then cooled to 60-65°C.

[0071] (Step 3) Sodium dodecyl sulfate was added to the sodium chloride aqueous solution containing fish essence to a final concentration of 1%, and the mixture was slowly stirred for 30 minutes.

[0072] (Step 4) After filtering the lysate of step 3 above through a filter having a pore size of 50 to 10 microns, ethanol is added to the filtrate to a final concentration of 50% to 80%, and allowed to stand for 20 minutes. After standing, the obtained precipitate is washed by adding 50% to 80% ethanol and slowly stirred. After washing, the precipitate is dehydrated in 95% ethanol and dried with a hot air dryer (55° C.) or a vacuum dryer (45° C., -0.09 to -0.1 MPa). Then, the dried product is completely dissolved in sterile distilled water to a final concentration of 1% to 3%.

[0073] (Step 5) The lysate obtained in step 4 was filtered through a 1 to 0.5 μm filter, and sodium chloride was added thereto to a final concentration of 1 M, followed by slow stirring at 85° C. for 16 hours.

[0074] (Step 6) Magnesium chloride was added to the lysate of step 5 to a final concentration of 20 mM, and the mixture was slowly stirred at 85°C for 3 hours and 30 minutes.

[0075] (Step 7) The lysate from step 6 above was sterile filtered through a filter with a pore size of 0.22 micrometers. 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 washed by adding 70% ethanol and slowly stirring for 16 hours.

[0076] (Step 8) After adding 95% ethanol to the washed precipitate in the above step 7, the mixture is slowly stirred for 10 minutes to dehydrate, and then dried using a hot air dryer (55°C) or a vacuum dryer (45°C, -0.09 to -0.1MPa) to prepare a high-purity DNA fragment mixture with a molecular weight of 1000 to 10000 KDa.

[0078] Example 2: Preparation of High-Purity DNA Fragment Mixture 2

[0079] 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.

[0081] Example 3: Preparation of High-Purity DNA Fragment Mixture 3

[0082] 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.

[0084] Comparative Example 1

[0085] A DNA fragment mixture was prepared in the same manner as in Example 1, except that distilled water was added in place of isopropanol in step 1 of Example 1.

[0087] Comparative Example 2

[0088] A 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.

[0090] Comparative Example 3

[0091] A DNA fragment mixture was prepared in the same manner as in Example 1, except that in step 6 of Example 1, heat treatment was performed at 100°C without adding magnesium chloride.

[0093] Comparative Example 4

[0094] A DNA fragment mixture was prepared in the same manner as in Example 1, except that 0.4% SDS was added in step 6 of Example 1 instead of magnesium chloride.

[0096] Comparative Example 5

[0097] A 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, but the pH was adjusted to 4 and treated at 85° C. for 20 minutes.

[0098] Experimental Example 1: Comparison of turbidity and purity according to fish sperm impurity removal methods

[0099] The turbidity and purity of the fish sperm impurity removal method according to the example in which isopropyl alcohol was added, the comparative example 1 in which distilled water was added, and the comparative example 2 in which nothing was added were compared.

[0100] PN (1 g) prepared based on each fish sperm impurity removal method was hydrated in 100 ml of sterile distilled water and the turbidity ( Figure 1 ). As a result, the transparency of the PN prepared by the method of adding isopropanol in the embodiment is the highest. 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 any substance are both opaque white, and in particular, the PN of Comparative Example 2 is the most opaque. Therefore, through the turbidity comparison, it is confirmed that the method of the present invention for preparing a DNA fragment mixture using isopropanol has the best effect of removing fish sperm impurities.

[0102] [Table 1] Absorbance Example 1 Comparative Example 1 Comparative Example 2 600nm 0.0034 0.0600 0.8300 280nm 0.5704 0.6704 0.3704 260nm 1.0340 1.1340 0.6034 230nm 0.4825 0.4925 0.2925 260 / 280 1.81 1.69 1.63 260 / 230 2.14 2.30 2.06

[0103] As shown in Table 1, as a result of comparing the purity of 1 g of PN prepared according to each impurity removal method after hydration in 100 ml of sterile distilled water, it was confirmed that 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.

[0105] Experimental Example 2: Comparison of PN yields according to fish sperm impurity removal methods

[0106] The PN yields of the fish sperm impurity removal methods according to the example in which isopropyl alcohol was added, the comparative example 1 in which distilled water was added, and the comparative example 2 in which nothing was added were compared.

[0107] [Table 2] Final PN yield Example 6％(12g) Comparative Example 1 4％(8g) Comparative Example 2 4％(8g)

[0108] As a result, as shown in Table 2, which shows the final PN yield according to the fish sperm impurity removal method, it is confirmed that the PN of the example prepared by the method of adding isopropanol has the highest yield of 6%.

[0110] Experimental Example 3: Comparison of molecular weight reduction effects according to nucleic acid molecular weight reduction methods

[0111] The molecular weight reduction effects of the nucleic acid molecular weight reduction methods according to Example 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.

[0112] Results, such as Figure 2As shown, when magnesium chloride was used, the best molecular weight reduction effect was observed, while in Comparative Examples 3 to 5, no molecular weight reduction effect was observed, or the observed effect was relatively low compared with the Examples. Therefore, it was confirmed that the method of preparing a DNA fragment mixture using magnesium chloride of the present invention has the best molecular weight reduction effect and can be used to produce a DNA fragment mixture with a molecular weight of 1000 to 10000 KDa.

[0114] Experimental Example 4: Comparison of molecular weight reduction effects according to magnesium chloride treatment time

[0115] In the above Experimental Example 3, it was confirmed that the magnesium chloride treatment method has the best DNA molecular weight reduction effect. Therefore, the inventors further confirmed the DNA molecular weight reduction effect according to the magnesium chloride treatment time. For comparison, magnesium chloride was treated for 1 hour (Example 3), 2 hours (Example 2) and 3 hours and 30 minutes (Example 3), respectively, and PN (HTL Company) was used as a control.

[0116] Results, such as Figure 3 and Figure 4 As shown, the longer the magnesium chloride treatment time, the better the DNA molecular weight reduction effect. In particular, the DNA molecular weight of the PN of Example 1 was analyzed to be 325 to 975 Kda, which is similar to the DNA molecular weight of the control PN.

[0118] Experimental Example 5: GPC analysis results based on magnesium chloride treatment time

[0119] According to Experimental Example 3, in order to more accurately compare the molecular weight, GPC (gel permeation chromatography) according to the magnesium chloride treatment time was performed. Specifically, the analysis was performed using a SHODEX OHpak SB-806M HQ 300mm column under the following conditions: column temperature 40°C, flow rate 1.0mL / min, RI detector, pressure 1.7MPa, injection volume 100ul. The sample was dissolved to 1% in pure water, diluted 25 times in mobile phase A, filtered through a 0.45μm filter, and then analyzed. The standards used at this time were Dextran standard 1 (2457000MW), Dextran standard 2 (1150000MW), and Dextran standard 3 (405700MW).

[0121] [Table 3] Minimum molecular weight Maximum molecular weight Average molecular weight Comparison 947 4312 2291 Example 3 253 6563 3959 Example 2 190 4921 3116 Example 1 766 4923 2014

[0122] As shown in Table 3 and Figures 5a to 5dAs shown, it was confirmed that as the magnesium chloride treatment time increased, the DNA molecular weight range decreased, and a DNA fragment mixture with a constant molecular weight was prepared, thereby inducing PN purification. In addition, it was confirmed that it had a similar average molecular weight compared to the control.

[0123] Experimental Example 6: Comparison of viscoelasticity between Example 1 and the control

[0124] The viscoelasticity of Example 1 and the control was compared. Specifically, the viscoelasticity of the sample was analyzed using a viscoelasticity device (MCR 92, manufacturer: Anton Paar), a rotor (measuring plate PP25 D: 25 mm, manufacturer: Anton Paar), a plunger rod and a scraper. At this time, a reference point was established using a viscosity standard (viscosity standard 1000, viscosity standard 5000-manufacturer: BROOKFIELD AMETE), and 1 ml of 1% PN (sample) dissolved in pure water was dispensed into the viscoelasticity device. Then, the storage modulus and loss modulus of each sample were measured at a total of 16 points, and the analysis was performed when the angular frequency ω (rad / s) was 1.

[0126] [Table 4]

[0127] 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 the viscoelasticity of Example 1 is superior.

Claims

1. A method for preparing a high-purity DNA fragment mixture, the method comprising: The following steps are involved: (Step 1) treating fish sperm with isopropyl alcohol to obtain fish sperm with impurities removed; (Step 2) treating the fish essence after the impurities are removed by adding it to a sodium chloride aqueous solution; (Step 3) adding an aqueous sodium dodecyl sulfate solution to an aqueous sodium chloride solution containing fish sperm to obtain a primary fish sperm lysate in which cells are decomposed and nucleic acids are extracted; (Step 4) precipitating the primary fish sperm lysate with ethanol and separating the precipitate to obtain a secondary fish sperm lysate; (Step 5) adding sodium chloride to the secondary fish sperm lysate and performing a first virus inactivation step at a high temperature of 80° C. to 100° C.; (Step 6) adding magnesium chloride to the virus-inactivated fish sperm lysate to obtain a mixture of DNA fragments with reduced nucleic acid molecular weight; and (Step 7) A step of performing secondary virus inactivation by treating the DNA fragment mixture whose nucleic acid molecular weight has been reduced with ethanol and then obtaining a precipitate.

2. The 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 sperm.

3. The method for preparing a high-purity DNA fragment mixture according to claim 1, wherein step 2 is to add a sodium chloride aqueous solution with a concentration of 20wt% to 50wt% to the fish sperm from which impurities have been removed, and treat it at a temperature of 90°C to 100°C for 1 hour to 4 hours, and then cool it.

4. The method for preparing a high-purity DNA fragment mixture according to claim 1, wherein step 3 comprises adding 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 treating at 10°C to 30°C for 20 minutes to 40 minutes.

5. The method for preparing a high-purity DNA fragment mixture according to claim 1, wherein step 4 is to precipitate the primary fish sperm lysate by adding ethanol until the final concentration is 50% to 80%, and separate the precipitate to obtain a secondary fish sperm lysate.

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

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

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

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

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

11. A high-purity DNA fragment mixture prepared by the method of claim 1.

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