Polydeoxyribonucleotides and process for their preparation

Abstract

The application provides a preparation method of polydeoxyribonucleotides, which comprises the following steps: obtaining salmon genome DNA, preparing amplified salmon DNA fragments by using multiple displacement amplification on the salmon genome DNA, and breaking the salmon DNA fragments to obtain polydeoxyribonucleotides. The application also provides polydeoxyribonucleotides, wherein the proportion of fragments with a length of less than or equal to 400 bp is greater than 90%, and the proportion of fragments with a length of greater than or equal to 1000 bp is less than 2%. The method provided by the application can synthesize a large amount of salmon sperm DNA by using synthetic biology technology and only by introducing a small amount of exogenous protein (Phi DNA polymerase). In addition, the salmon sperm DNA synthesized by the application is not full-length genome DNA (gDNA) but long fragment DNA with a fragment size of 12 kb to 100 kb; and a large amount of PDRN samples with ideal molecular weight can be obtained in a short time by using a composite physical crushing method and a biological treatment means.

Description

Technical Field

[0001] This application belongs to the field of bioengineering, specifically relating to a polydeoxyribonucleotide and a method for its preparation. Background Technology

[0002] Polydeoxyribonucleotides (PDRN) are a class of naturally occurring bioactive substances derived from the testes or semen of salmon, trout, or chum salmon. Essentially, they are deoxyribonucleotide conjugates with a molecular weight ranging from 50 to 1,500 kDa. Due to the high similarity (≥95%) between the salmon genome and the human genome in terms of base structure and composition, rigorously purified PDRN exhibits no immunogenicity and high safety and stability. In vitro and in vivo experiments have shown that adenosine A2A receptors on the cell surface, by recognizing the adenosine receptor agonist PDRN, can regulate inflammation, reduce cellular oxygen consumption, improve local tissue ischemia, and promote cell growth and angiogenesis. Furthermore, PDRN can provide nucleosides and nucleotides for cell growth by participating in the "rescue pathway," extending cell lifespan and improving cell condition. In summary, PDRN, as a novel functional raw material, has functions such as tissue repair, wound healing promotion, and anti-aging, and is currently widely used in pharmaceuticals, cosmetics, food, and health products.

[0003] Fish testes and semen, rich in DNA, are ideal raw materials for PDRN extraction. However, due to unforeseen risks such as heavy metal contamination, gene mutations, and chromosomal aberrations caused by oil spills, nuclear wastewater discharges, and ship pollution, direct PDRN extraction from salmon testes or semen is no longer the optimal choice. Furthermore, as a vital component of the ecosystem, deep-sea salmon populations are susceptible to degradation and extinction due to modern fishing practices. The 21st century is an era of explosive growth in biotechnology, with synthetic biology, through engineered strategies, enabling the targeted design and synthesis of standardized biological components, the construction of specific gene circuits, and the assembly of artificial life systems with specific functions. This allows for the targeted artificial synthesis of various raw materials, drugs, and bioenergy. The production process of multiple PDRNs mainly consists of two major steps: DNA extraction and DNA fragmentation. Existing industrial DNA extraction methods include alcohol precipitation, salting out, and column chromatography, while DNA fragmentation methods include physical fragmentation (ultrasonic fragmentation) and biological fragmentation (enzymatic digestion). For example, patent CN107287186A discloses a method for separating polynucleotides from fish semen, which purifies salmon DNA through salt precipitation and obtains PDRN through physical fragmentation; patent CN106031709B (authorized by Huiwen) discloses a method for preparing fish sperm DNA-NA, protamine extract, and related materials, which uses column chromatography to obtain a high concentration of fish sperm DNA-NA extract; and patent CN112315836A discloses a method for preparing high-efficiency topical PDRN, which obtains high-purity DNA through alcohol precipitation and then obtains high-quality PDRN through restriction endonuclease treatment. Because DNA is extracted directly from animal cells, industrial DNA extraction processes easily result in a large amount of protein residue. The single fragmentation process in PDRN production not only has the disadvantages of high power consumption, high cost, and long reaction time, but also does not always yield the ideal molecular weight. For example, patent CN107287186A did not measure the amount of residual protein in the sample and lacked key evidence to characterize the molecular weight and distribution range; patent CN106031709B obtained DNA samples with a residual protein content of 2.5-5% and did not mention the molecular weight and distribution range; although patent CN112315836A had a residual protein content of less than 1% and a relatively ideal PDRN distribution range, the use of a single enzyme digestion process (incubation at 37°C for 16 hours) in the DNA fragmentation process was not only costly but also had a long reaction time. Summary of the Invention

[0004] To address the problems existing in the prior art, this application provides a polydeoxyribonucleotide and a method for its preparation.

[0005] Specifically, this application relates to the following aspects:

[0006] 1. A method for preparing polydeoxyribonucleotides, characterized in that the preparation method comprises the following steps:

[0007] Obtain salmon genomic DNA,

[0008] Amplified salmon DNA fragments were prepared by multiple substitution amplification of salmon genomic DNA. The salmon DNA fragments were then broken down to obtain polydeoxyribonucleotides.

[0009] 2. The preparation method according to item 1, characterized in that the preparation of the amplified salmon DNA fragment by multiple substitution amplification of salmon genomic DNA includes the following steps:

[0010] The obtained salmon genomic DNA was processed to obtain a single-stranded salmon genomic DNA template.

[0011] Neutralize the template,

[0012] Random primers were used for multiple displacement amplification in a PCR system containing neutralized template, Phi29 DNA polymerase, and dNTPs.

[0013] The Phi29 DNA polymerase was inactivated to obtain the amplified salmon DNA fragment.

[0014] 3. The preparation method according to item 2, characterized in that the concentration of the random primer in the PCR system is 0.2-1.0 μM.

[0015] 4. The preparation method according to item 2, characterized in that the multiple displacement amplification time is 2-4 hours.

[0016] 5. The preparation method according to item 1, characterized in that the breaking includes ultrasonic treatment and / or enzymatic digestion, preferably, the breaking includes first performing ultrasonic treatment and then performing enzymatic digestion.

[0017] 6. The preparation method according to item 5, characterized in that the ultrasonic power of the ultrasonic treatment is 200-400W, the duty cycle is 1:0.5-2, and the ultrasonic treatment time is 10-60min.

[0018] 7. The preparation method according to item 5, characterized in that the enzymatic digestion treatment uses a restriction endonuclease Sau 3AI with a concentration of 50-100 U / ml and is kept at a constant temperature of 37-40℃ for 1-2 hours.

[0019] 8. The preparation method according to item 1, characterized in that the preparation method further includes the step of breaking the salmon DNA fragment, removing the protein, and precipitating to obtain the salmon DNA fragment.

[0020] 9. A polydeoxyribonucleotide, characterized in that fragments with a length of 400 bp or less account for more than 80%, preferably more than 90%.

[0021] Further optimization is to ensure that fragments with a length of 200bp or less account for more than 40%, preferably more than 50%.

[0022] Further optimization is to ensure that fragments with a length of 600bp or less account for more than 90%, preferably more than 95%.

[0023] Further, it is preferred that the proportion of fragments with a length greater than or equal to 1000bp be less than 10%, and preferably less than 2%.

[0024] 10. The polydeoxyribonucleotide according to item 9, characterized in that the polydeoxyribonucleotide is prepared by any one of items 1-8.

[0025] To address the shortcomings of protein residue and single-fragmentation treatments in industrial DNA extraction, this application provides a method for isothermal amplification and biosynthesis of polydeoxyribonucleotides (PDRNs). Using synthetic biology techniques, only a small amount of exogenous protein (Phi DNA polymerase) is needed to synthesize large quantities of salmon sperm DNA. Furthermore, the salmon sperm DNA synthesized in this application is not full-length genome DNA (gDNA) but rather a long DNA fragment with a size ranging from 12kb to 100kb; through a combination of physical fragmentation methods and biological processing techniques, a large quantity of PDRN samples with ideal molecular weights can be obtained in a short time. Attached Figure Description

[0026] Figure 1 Gel electrophoresis images of PDRN under different reaction conditions are shown. A shows gel electrophoresis images with different primer concentrations (0.2–1.0 μM), and B shows gel electrophoresis images with different reaction times (2–4 h).

[0027] Figure 2 The images show gel electrophoresis patterns of PDRN under different sonication conditions. A shows gel electrophoresis patterns with different sonication times (10–60 min), B shows gel electrophoresis patterns with different sonication powers (200–400 W), and C shows gel electrophoresis patterns with different duty cycles (1–2:1–2).

[0028] Figure 3 Fragment distribution results of PDRN prepared for analysis using a nucleic acid / protein analyzer;

[0029] Figure 4 Fragment distribution results of PDRN prepared in a comparative sample, as determined by a nucleic acid / protein analyzer;

[0030] Figure 5 The fragment distribution results of PDRN prepared for gel electrophoresis determination in the examples and comparative examples are shown. Detailed Implementation

[0031] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0032] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0033] This application provides a method for preparing polydeoxyribonucleotides, the method comprising the following steps:

[0034] Step 1: Obtain salmon genomic DNA.

[0035] Step 2: Amplified salmon DNA fragments were prepared by multiple substitution amplification of salmon genomic DNA.

[0036] Step 3: Break down the salmon DNA fragment to obtain polydeoxyribonucleotides.

[0037] In step one, salmon genomic DNA can be extracted from salmon using existing techniques. For example, pure salmon genomic DNA (gDNA) can be obtained through the traditional phenol-chloroform extraction method or a kit. The traditional phenol-chloroform extraction protocol uses phenol / chloroform to separate the organic and aqueous phases, thereby extracting nucleic acids. Although phenol-chloroform extraction can yield a considerable amount of gDNA, it is unsuitable for large-scale industrial production due to the use of toxic and harmful reagents. While commercially available kits can obtain relatively pure gDNA, the yield is low and the cost is high, making industrial-scale production difficult. Therefore, this application uses a small amount of gDNA (salmon gDNA) extracted by a kit as a template, and a large amount of salmon gDNA can be obtained through multiple displacement amplification technology, without involving the use of any toxic or harmful reagents during the reaction.

[0038] In step two, the multiple displacement amplification (MDA) technique used is currently the most commonly used whole genome amplification method. Under constant temperature conditions of 30°C, random primers bind to template DNA through random annealing. Under the action of high-fidelity, strong strand displacement activity Phi29 DNA polymerase, a large number of amplified fragments of 12kb to 100kb can be synthesized in a short time.

[0039] Furthermore, the preparation of the amplified salmon DNA fragment by multiple displacement amplification may include the following steps: processing the obtained salmon genomic DNA to obtain a single-stranded salmon genomic DNA template; neutralizing the template; performing multiple displacement amplification with random primers in a PCR system containing the neutralized template, Phi29 DNA polymerase, and dNTPs; and inactivating the Phi29 DNA polymerase to obtain the amplified salmon DNA fragment.

[0040] The process of converting DNA from double strands to single strands can be performed using any denaturation method known in the art.

[0041] Phi29 DNA polymerase is a thermophilic DNA polymerase cloned from Bacillus subtilis phage Phi29. It was expressed in Escherichia coli and then purified and isolated multiple times.

[0042] Phi29 DNA polymerase possesses unique strand substitution and continuous synthesis characteristics, enabling the continuous synthesis of DNA fragments up to 70 kb in length. Furthermore, this enzyme exhibits 3'→5' exonuclease proofreading capabilities, resulting in highly faithful synthesized DNA fragments. Due to the enzyme's strong exonuclease activity, modifying the 3' end of the primers during synthesis to reduce the cleavage effect of the exonuclease activity is crucial.

[0043] The concentration of random primers and the amplification time can be adjusted during multiple substitution amplification.

[0044] In one specific embodiment, the concentration of the random primers in the PCR system is 0.2-1.0 μM, for example, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1.0 μM.

[0045] In one specific implementation, the multiple substitution amplification time is 2-4 hours, for example, 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours.

[0046] In step three, the salmon DNA fragment can be processed by methods known in the art, such as sonication, enzyme digestion, or a combination of sonication and enzyme digestion.

[0047] In one specific implementation, the disruption includes first performing sonication followed by enzymatic digestion.

[0048] The ultrasonic treatment can be achieved using conventional ultrasonic equipment. Specifically, the DNA fragmentation effect can be controlled by adjusting the ultrasonic power, duty cycle, and treatment time. In one specific embodiment, the ultrasonic power for the ultrasonic treatment is 200-400W, for example, 200W, 250W, 300W, 350W, or 400W; the duty cycle is 1:0.5-2, for example, 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5, 1:1.8, or 1:2; and the ultrasonic treatment time is 10-60 minutes, for example, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes.

[0049] In one specific embodiment, the enzyme digestion treatment uses a restriction endonuclease Sau 3AI with a concentration of 50-100 U / ml and is kept at a constant temperature of 37-40°C for 1-2 hours.

[0050] Furthermore, the preparation method further includes the steps of breaking down the salmon DNA fragment, removing proteins, and precipitating the fragment to obtain the salmon DNA fragment. The method for removing proteins is not limited and can be any method known in the art; in one specific embodiment, proteins are removed by protease. The precipitation of the salmon DNA fragment can be obtained by isopropanol precipitation, washing, and freeze-drying.

[0051] This application also provides a PDRN, wherein the proportion of fragments with a length of 400 bp or less in the polydeoxyribonucleotide is greater than 80%, preferably greater than 90%.

[0052] Furthermore, this application also provides a PDRN, wherein the proportion of fragments with a length of 400 bp or less in the polydeoxyribonucleotide is greater than 80%, preferably greater than 90%, and the proportion of fragments with a length of 200 bp or less is greater than 40%, preferably greater than 50%.

[0053] Furthermore, this application also provides a PDRN, wherein the proportion of fragments with a length of less than or equal to 400 bp in the polydeoxyribonucleotide is greater than 80%, preferably greater than 90%, and the proportion of fragments with a length of less than or equal to 200 bp is greater than 40%, preferably greater than 50%, and the proportion of fragments with a length of less than or equal to 600 bp is greater than 90%, preferably greater than 95%.

[0054] Furthermore, this application also provides a PDRN, wherein the proportion of fragments with a length of 400 bp or less is greater than 80%, preferably greater than 90%, and the proportion of fragments with a length of 200 bp or less is greater than 40%, preferably greater than 50%, and the proportion of fragments with a length of 600 bp or less is greater than 90%, preferably greater than 95%, and the proportion of fragments with a length of 1000 bp or more is less than 10%, preferably less than 2%.

[0055] According to the recommendations of BR PHARM, a leading South Korean PDRN raw material company, the appropriate PDRN fragment length is 700 kDa for medical device grade, 350–500 kDa for pharmaceutical grade, and 40–60 kDa for cosmetic and food grade. Therefore, the PDRN prepared in this application can be used in pharmaceuticals or food, and has a wide range of applications.

[0056] Furthermore, the PDRN can be prepared by the above-described preparation method.

[0057] The preparation method described in this application not only overcomes the shortcomings of existing technologies, such as poor sustainability and environmental unfriendliness, but also requires only a trace amount of salmon DNA to produce large quantities of PDRN, significantly reducing the amount of exogenous protein introduced. Existing production processes typically extract PDRN from the testes of adult male salmon. The average weight of a normal adult male Atlantic salmon is approximately 4.5 kg, and the testes weigh approximately 300 g. The nucleic acid content in salmon testicular cells is approximately 7%, therefore, one male Atlantic salmon can provide less than 15 g of PDRN raw material (including nucleic acid loss during processing). In contrast, this application achieves 125 μg of PDRN raw material using only 50 ng of template. Therefore, under the same conditions, this application can produce approximately 37,500 g of PDRN raw material using a template provided by one male Atlantic salmon, which is 2,500 times that of conventional methods. Thus, the protein residue in this application's method is only 1 / 2,500 of that in conventional methods. Furthermore, this application's method can use only pure gDNA, ensuring the safety of PDRN.

[0058] Example

[0059] Example 1

[0060] The optimization of the Multiple Displacement Amplification (MDA) method follows these steps:

[0061] Step 1: Prepare reagents by thawing the required reagents in advance.

[0062] Thaw the Phi29 DNA polymerase reaction buffer at room temperature. The Phi29 DNA polymerase reaction buffer contains 40 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, and 5 mM (NH4)2SO4.

[0063] Step 2: Denaturation treatment of salmon DNA template, converting double-stranded DNA template into single-stranded DNA:

[0064] Add 2.5 μL of salmon sperm gDNA (D8030, Solarbio) to a microcentrifuge tube. The gDNA concentration is ~50 ng / μL. Denaturation treatment: Add 2 μL of denaturation buffer to the DNA sample, mix well by pipetting, and incubate at room temperature for 2.5-3 min (denaturation buffer: 1 μL of 0.5M EDTA (pH 8); 4 μL of 5M KOH; 35 μL of nuclease-free water).

[0065] Step 3: Neutralize the DNA template and adjust the pH of the solution to prepare for amplification.

[0066] Add 2 μL of neutralization solution (neutralization solution: 400 mM HCl and 600 mM Tris-HCl (pH 7.5)) to the denatured DNA template described in step 2 and mix well by pipetting.

[0067] Step 4: Multiple displacement amplification, preparation of salmon DNA samples:

[0068] Prepare the mixing system according to the following ratio (as shown in Table 1): 4 μL Phi29 DNA polymerase reaction buffer, 4 μL 10 mM dNTPs, 2 μL random primers with final concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0 μM respectively, 0.8 μL 10 mg / mL BSA, 0.5 μL Phi29 DNA polymerase, 31.2 μL water, for a total volume of 42.5 μL; take 2.5 μL of the denatured DNA template described in step 3, add it to the 42.5 μL mixing system, and incubate at 30°C in a PCR instrument or other constant temperature equipment for 2 or 4 h.

[0069] Table 1

[0070]

[0071] Step 5: DNA polymerase inactivation, terminating the reaction.

[0072] At the end of the incubation, heat the DNA sample to 65°C for 10 minutes, then cool the sample to 4°C for storage.

[0073] Step 6: Agarose gel electrophoresis verification

[0074] Weigh 1g of agarose powder into a 250mL Erlenmeyer flask, add 99mL of 1×TAE buffer to prepare a 1% agarose gel. Detect amplification quality using the 1% agarose gel at 120V for approximately 30 minutes. Remove the gel and observe the results using UV imaging on a gel imaging system.

[0075] The results are as follows Figure 1 As shown, when the MDA primer concentration is 0.6 μM and the reaction time is 4 h, clear and bright bands are displayed on the agarose gel electrophoresis image.

[0076] Example 2

[0077] The method for ultrasonic fragmentation of gDNA was optimized, following these steps:

[0078] Step 1: Reagent preparation. Thaw the required reagents in advance.

[0079] Thaw the Phi29 DNA polymerase reaction buffer at room temperature. The Phi29 DNA polymerase reaction buffer contains 40 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, and 5 mM (NH4)2SO4.

[0080] Step 2: Denaturation treatment of salmon DNA template, converting double-stranded DNA template into single-stranded DNA:

[0081] Add 2.5 μL of salmon sperm gDNA to a microcentrifuge tube (gDNA concentration: ~50 ng / μL); denaturation treatment: add 2 μL of denaturation buffer to the DNA sample, mix well by pipetting, and incubate at room temperature for 2.5-3 min (denaturation buffer: 1 μL of 0.5 M EDTA (pH 8); 4 μL of 5 M KOH; 35 μL of nuclease-free water);

[0082] Step 3: Neutralize the DNA template and adjust the pH of the solution to prepare for amplification.

[0083] Add 2 μL of neutralization solution to the denatured DNA template described in step 2 and mix well by pipetting (neutralization solution: 400 mM HCl and 600 mM Tris-HCl (pH 7.5));

[0084] Step 4: Multiple displacement amplification, preparation of salmon DNA samples:

[0085] Prepare the mixing system according to the following ratio (as shown in Table 2): 4 μL Phi29 DNA polymerase reaction buffer, 4 μL 10 mM dNTPs, 2 μL random primers (final concentration 0.6 μM), 0.8 μL 10 mg / mL BSA, 0.5 μL Phi29 DNA polymerase, 31.2 μL water, total volume 42.5 μL; take 2.5 μL of the denatured DNA template described in step 3, add it to the 42.5 μL mixing system, and incubate at 30°C in a PCR instrument or other constant temperature equipment for 4 h;

[0086] Table 2

[0087]

[0088] Step 5: DNA polymerase inactivation, terminating the reaction.

[0089] At the end of the incubation, heat the DNA sample to 65°C for 10 minutes, then cool the sample to 4°C for storage.

[0090] Step 6: Ultrasonic treatment of salmon DNA:

[0091] Ultrasonic treatment: The DNA sample prepared in step 5 was subjected to ultrasonic disruption. The ultrasonic power, duty cycle, and ultrasonic treatment time were optimized. The ultrasonic treatment time was 10 min, 20 min, 30 min, 40 min, and 50 min, the ultrasonic power was 200 W, 250 W, 300 W, 350 W, and 400 W, and the duty cycle was 2:1, 2:2, 1:1, and 1:2.

[0092] Step 7: Agarose gel electrophoresis verification

[0093] Weigh 1g of agarose powder into a 250mL Erlenmeyer flask, add 99mL of 1×TAE buffer to prepare a 1% agarose gel. Detect amplification quality using the 1% agarose gel at 120V for approximately 30 minutes. Remove the gel and observe the results using UV imaging on a gel imaging system.

[0094] This embodiment uses the amplification products of MDA as samples to optimize the ultrasound time, power, and duty cycle. The results are as follows: Figure 2 As shown, when the sonication time is 30 min, the sonication power is 250 W, and the duty cycle is 2:1, the agarose gel electrophoresis image shows a relatively good disruption effect.

[0095] Example 3

[0096] A method for isothermal amplification of polydeoxyribonucleotides for biosynthesis, comprising the following steps:

[0097] Step 1: Reagent preparation. Thaw the required reagents in advance.

[0098] Thaw the Phi29 DNA polymerase reaction buffer at room temperature. The Phi29 DNA polymerase reaction buffer contains 40 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, and 5 mM (NH4)2SO4.

[0099] Step 2: Denaturation treatment of salmon DNA template, converting double-stranded DNA template into single-stranded DNA:

[0100] Add 2.5 μL of salmon sperm gDNA to a microcentrifuge tube (gDNA concentration: ~50 ng / μL); denaturation treatment: add 2 μL of denaturation buffer to the DNA sample, mix well by pipetting, and incubate at room temperature for 2.5-3 min (denaturation buffer: 1 μL of 0.5 M EDTA (pH 8); 4 μL of 5 M KOH; 35 μL of nuclease-free water);

[0101] Step 3: Neutralize the DNA template and adjust the pH of the solution to prepare for amplification.

[0102] Add 2 μL of neutralization solution to the denatured DNA template described in step 2 and mix well by pipetting (neutralization solution: 400 mM HCl and 600 mM Tris-HCl (pH 7.5));

[0103] Step 4: Multiple displacement amplification, preparation of salmon DNA samples:

[0104] Prepare the mixing system according to the following ratio (as shown in Table 2): 4 μL Phi29 DNA polymerase reaction buffer, 4 μL 10 mM dNTPs, 2 μL random primers (0.6 μM), 0.8 μL 10 mg / mL BSA, 0.5 μL Phi29 DNA polymerase, 31.2 μL water, total volume 42.5 μL; take 2.5 μL of the denatured DNA template described in step 3, add it to the 42.5 μL mixing system, and incubate at 30°C in a PCR instrument or other constant temperature equipment for 4 h;

[0105] Step 5: DNA polymerase inactivation, terminating the reaction.

[0106] At the end of the incubation, heat the DNA sample to 65°C for 10 minutes, then cool the sample to 4°C for storage.

[0107] Step 6: Combined ultrasonication and enzyme digestion treatment of salmon DNA to obtain PDRN samples.

[0108] (1) Ultrasonic treatment: The DNA sample prepared in step 5 is subjected to ultrasonic disruption treatment. The ultrasonic power is 250W, the duty cycle is 2:1, and the ultrasonic treatment lasts for 30 minutes.

[0109] (2) Enzyme digestion: Add 50-100 U of restriction endonuclease Sau 3AI to the DNA solution prepared above and treat at 37℃ for 2 hours;

[0110] (3) Protein removal: Add 90-100 mM NaCl, 0.9-1% SDS, and 0.04-0.05 mg / mL proteinase K to the DNA solution prepared above, keep it at 50°C for 3 h, centrifuge to remove the precipitate, and collect the supernatant.

[0111] (4) Add an equal volume of isopropanol to the supernatant obtained above and precipitate for 30 min (room temperature). Centrifuge and discard the supernatant. Wash the precipitate twice with 70% or 75% ethanol. Freeze-dry the precipitate to obtain a white solid, which is the PDRN sample.

[0112] Comparative Example

[0113] Comparative Example 1 uses the method described in CN107287186A in the background art, which uses an extraction method to extract PDRN from salmon testes. The extraction process involves a -80°C treatment, and the processing method is complicated and not suitable for large-scale industrial production.

[0114] Comparative Example 2 uses the method described in CN112315836A in the background art, which also uses an extraction method to extract PDRN raw material from salmon testes, with an extraction yield of ~15%.

[0115] Calculations show that the PDRN sample prepared in Example 3 can synthesize 125 μg of PDRN sample using 0.05 μg salmon DNA as a template.

[0116] The fragment distribution of PDRN samples prepared in Example 3 and Comparative Example 2 was analyzed using the Qsep fully automated nucleic acid / protein analyzer, where the results of Example 3 are as follows: Figure 3 As shown, the results of Comparative Example 2 are as follows: Figure 4 As shown. The fragment distribution of the PDRN samples prepared in Example 3 and Comparative Example 2 was determined using agarose gel electrophoresis, and the results are as follows. Figure 5 As shown, samples 1-4 (four parallels) are PDRN samples obtained after ethanol washing in step 6 of Example 3; samples 4-8 (four parallels) are PDRN samples reconstituted after lyophilization in step 6 of Example 3; samples 7-8 are PDRN samples prepared in Comparative Example 2 (two parallels); and M is the molecular weight standard. Figure 3 and Figure 4 The results show that in the PDRN samples prepared in this application, fragments with a base length of 24–400 bp account for more than 90%, while fragments exceeding 1000 bp account for only about 1%. In contrast, in the PDRN samples prepared in Comparative Example 2, fragments below 400 bp account for only 34.3%, while fragments exceeding 1000 bp account for 49.8%.

[0117] The properties, solubility, and protein content of the PDRN samples prepared in Example 3, Comparative Example 1, and Comparative Example 2 were further tested, and the results are shown in Table 3. The protein content was determined using the Coomassie Brilliant Blue method. A comparison of the detection results between the examples and the comparative examples is shown in Table 3.

[0118] Table 3

[0119]

[0120]

Claims

1. A method for preparing polydeoxyribonucleotides, characterized in that, The preparation method includes the following steps: Obtain salmon genomic DNA, Amplified salmon DNA fragments were prepared by multiple substitution amplification of salmon genomic DNA. The salmon DNA fragment was broken down, proteins were removed, and precipitated to obtain polydeoxyribonucleotides; The preparation of amplified salmon DNA fragments using multiple substitution amplification of salmon genomic DNA includes the following steps: The obtained salmon genomic DNA was processed to obtain a single-stranded salmon genomic DNA template. Neutralize the template, Random primers were used for multiple displacement amplification in a PCR system containing neutralized template, Phi29 DNA polymerase, and dNTPs. The Phi29 DNA polymerase was inactivated to obtain the amplified salmon DNA fragment. The disruption process involves first sonication, followed by enzymatic digestion. The concentration of the random primers in the PCR system was 0.2-1.0 μM, the ultrasonic power for the ultrasonic treatment was 200-400 W, the duty cycle was 1:0.5-2, and the ultrasonic treatment time was 10-60 min. The enzyme digestion treatment uses a restriction endonuclease at a concentration of 50-100 U / ml and is carried out at a constant temperature of 37-40℃ for 1-2 hours.

2. The preparation method according to claim 1, characterized in that, The time for multiple substitution amplification is 2-4 hours.

3. The preparation method according to claim 1, characterized in that, The restriction endonuclease includes Sau 3AI.

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