RU2268730C1 - Method for production of heterogeneous deoxyribonucleic acid (dna) sodium salt - Google Patents

Abstract

FIELD: medicine, drug production.

SUBSTANCE: claimed method for production of heterogeneous deoxyribonucleic acid (DNA) sodium salt from animal male gonads and/or sperm thereof includes grinding and homogenization of starting raw materials in detergent solution, solution treatment at elevated temperature with simultaneous stirring, liquid phase separation by filtering and product precipitation with alcohol. Homogenization is carried out in 1-4 % sodium dodecyl sulfate solution in distilled water at 35-60°C with continuous stirring for 1.5-4 h followed by addition of coagulation agent, namely alkali or earth alkali metal permanganate to produce coagulation agent concentration in solution of 0.0005-0.03 M. Obtained solution is stirred at 35-60°C for 25-90 min, than ionic force is increased up to 1-3 M, solution is heated up to 60-62°C and stirred at abovementioned temperature for 1-3 h. Further contaminant precipitate obtained in coagulation process is separated by filtration thought filter having pore size from 1 to 25 mum. Filtered solution of non-fragmented (high molecular) DNA is cooled to 0-20°C, alcohol is added to obtain finished concentration of 27043.5 vol. %, Then obtained solution is agitated and deposited with alcohol preparation is filtered and separated, washed with 96 % ethanol and stored under ethanol. Obtained DNA preparation is dissolved in 0.01-4.5 M NaCl aqueous solution at 50°C or less; obtained solution is passed through high pressure homogenizer under pressure up to 2000 kg/cm², obtained fragmented DNA is treated with alcohol wherein deposited with alcohol fermented sodium DNA is centrifuged and separated. Separated precipitate is rewashed with alcohol and dried in desiccator at 50°C or less to produce powder.

EFFECT: simplified technology for DNA sodium salt production with increased yield without losses of quality.

1 ex, 3 tbl

Description

The invention relates to medicine, specifically to a method for producing a medicine, namely the sodium salt of heterogeneous deoxyribonucleic acid (DNA), which has found wide application in medical practice.

DNA as a biochemical substance in laboratory practice stands out for more than half a century. By the 60s of the last century, the main methods and approaches to the isolation process were formulated ["Nucleic Acids", ed. I. B. Zbarsky, M., Mir, 1966]. In almost all the techniques described by then, the extraction process was reduced to the following main steps:

1. Homogenization of raw materials and extraction of water-soluble impurities in aqueous solutions at low ionic strength, obtaining the source material for DNA extraction by precipitation in a centrifuge.

2. DNA extraction in the presence of a detergent.

3. Removal of proteins and other impurities soluble in the detergent solution by creating conditions under which they become poorly soluble (phenol treatment, salting out, etc.).

4. Removing aggregated impurities by centrifugation or filtration.

5. Precipitation of DNA by adding a high concentration of alcohols (ethyl, isopropyl, etc.) to the solution.

To achieve a higher degree of DNA purification, a repetition of paragraphs 1, 3, 4 was used.

Although the developed approaches and methods were widely used in laboratory practice, they had a number of drawbacks that made their application on an industrial scale labor-intensive and expensive. One of the main disadvantages of these methods was the use of expensive centrifuges with limited volumes.

Since the 90s, DNA began to be used as a biologically active product in medicine and the cosmetics industry. This imposed additional specific requirements on the preparations released industrially. In particular, a high degree of purification and relatively low molecular weights were required, i.e. there was a need for DNA fragmentation. In addition, there was a need for long-term storage of the resulting sterile DNA preparations with the preservation of their physico-chemical properties. Most of the patents on DNA isolation methods registered to date are aimed at solving the above problems of industrial production of DNA preparations, namely, reducing production costs, speeding up and simplifying production, and increasing the purity and shelf life of the resulting preparations.

In particular, a method is known for producing a native sterile 0.1-0.8% DNA solution with a molecular weight of 280-400 kDa (kiloDaltons) in the range of molecular weights of ± 30 kDa in a 0.09-0.11% solution sodium chloride used as a medicine (see patent RU No. 2039564, A 61 K 35/56, 07.20.1995).

This method allows you to select a DNA preparation with a relatively narrow distribution of molecular masses of molecules and reduce the proportion of molecules with sticky ends. The latter requirement leads to a decrease in their aggregation ability and to an increase in the shelf life of a sterile solution of native non-aggregated DNA. However, this production method is rather complicated and time-consuming, since it requires the use of ultrasonic treatment and the use of membrane technologies.

There is a known method of producing DNA, in which to accelerate the process and eliminate centrifugation while removing coagulated impurities (the above item 4), the DNA extract is pre-treated with ultrasound to reduce the viscosity of the solution, and then kieselguhr is added to the solution, which helps to form a precipitate convenient for filtration. This procedure allows to significantly increase the filtration rate (see patent RU No. 2017496, A 61 K 35/60, C 07 H 21/04 12/28/1990). However, this method also requires the use of a rather slow process of ultrasonic treatment and the use of membrane technologies.

The closest to the invention in technical essence and the achieved result is a method for producing the sodium salt of heterogeneous deoxyribonucleic acid (DNA) from the testes of animals and their sperm, including grinding and homogenization of the feedstock in a detergent solution, DNA extraction at elevated temperature, separation of the liquid phase by filtration and precipitation the product with alcohol (see patent RU No. 20055724, 01/15/1994).

This method allows to increase productivity and obtain a higher DNA yield with characteristics that are necessary for a significant expansion of its field of application (limited reduction in molecular weight while maintaining the native structure and high quality of purification). However, this method of obtaining sodium salt of DNA is quite complicated and requires strict adherence to the technology for its preparation.

The problem to which the present invention is directed, is to simplify the technology for producing sodium salt of DNA and increase the yield of the final product while maintaining its qualitative characteristics.

This problem is solved due to the fact that the method of obtaining the sodium salt of heterogeneous deoxyribonucleic acid (DNA) from the testes of animals and / or their sperm includes grinding and homogenizing the feedstock in a detergent solution, processing the solution at an elevated temperature while stirring the solution, and separating the liquid phase by filtration and precipitation of the product with alcohol, while homogenization is carried out in a 1-4% solution of sodium dodecyl sulfate in distilled water at a temperature of 35-60 ° C and constant stirring solution for 1.5-4 hours, after which a coagulant — an alkaline or alkaline earth metal permanganate — is added to the solution to obtain a coagulant concentration in the solution of 0.0005-0.03 M; the resulting solution is stirred at a solution temperature of 35-60 ° C for 25-90 min, then the ionic strength of the solution is increased to 1-3 M by adding salt to the solution (hydrochloric or sulfuric acid salts are most convenient), and the solution is heated to 60-62 ° C and solution 1 is mixed at the temperature indicated above -3 h, then the precipitate of impurities obtained during coagulation is separated filtering through a filter with pores ranging in size from 1 to 25 μm, the filtered solution of unfragmented (high molecular weight) DNA is cooled to a temperature of 0-20 ° C, then alcohol is added to the solution to a final concentration of 27-43.5 vol%, and then the resulting solution is stirred and the resulting alcohol-precipitated preparation was separated by filtration, washed with 96% ethyl alcohol and stored under ethyl alcohol.

In addition, a preparation with a given molecular weight in the range from 150 to 1,500 kDa can be obtained in the form of a powder, for which purpose the DNA preparation obtained as described above is dissolved in an aqueous NaCl solution at a concentration of 0.01-4.5 M at a temperature not exceeding 50 ° C, the resulting solution is passed through a high-pressure homogenizer at a pressure of up to 2000 kg / cm ² , the resulting fragmented DNA is treated with alcohol to obtain an alcohol concentration of 40 to 90%, and the fragmented sodium precipitated in alcohol is separated by centrifugation, after whereby the separated precipitate is washed again with alcohol and dried in an oven at a temperature not exceeding 50 ° C to obtain a powder.

One of the main problems with DNA isolation is a quick and cheap way to remove coagulated protein from a DNA extract (points 3 and 4 above). The problem is aggravated by the high viscosity of the solution at this stage, which complicates both the filtration of the solution and its centrifugation. To some extent, this problem was previously solved by fragmenting DNA in the early stages of the process (lowering the viscosity of the solution) and / or by adding kieselguhr to form a precipitate, which made it possible to increase the filtration rate. To overcome this problem and significantly increase the filtration rate at this stage even without preliminary fragmentation of the molecules, we were able to introduce a specific coagulator — permanganate of an alkali or alkaline earth metal at an elevated temperature, followed by an increase in the ionic strength of the solution. This leads to a significant increase in the size and stiffness of particles of coagulated material (denatured protein, detergent and other impurities) and a significant increase in the filtration rate of unfragmented DNA solution on virtually any filter with pore sizes less than 25 μm. In real production, this stage is as follows. After homogenizing the raw material and extracting the DNA in a 1-4% solution of sodium dodecyl sulfate in distilled water at a temperature of 35-60 ° C and constantly stirring the solution for at least 1.5 hours, alkaline or alkaline-earth metal permanganate is added to the concentration to a concentration of 0, 0005-0.03 M depending on the raw materials used. The coagulation process lasts 25-90 minutes. A subsequent increase in the ionic strength of the solution to 1.0-3.0 M, heating the solution to 60-62 ° C and stirring it for 1-3 hours at the above temperature leads to a decrease in the solubility of detergent, denatured proteins, and other impurities (salting out) and the formation of large coarse particles from them, which can easily be removed by filtration through a filter with pore sizes from 1 to 25 microns. The filtered transparent and unpainted solution of unfragmented (high molecular weight) DNA is cooled to a temperature of 0-20 ° C.

A further task is to remove residual protein, RNA, and other water-soluble impurities. In classical extraction methods, this problem was mainly solved by preliminary extraction of water-soluble impurities at a low ionic strength of the solution (paragraph 1). In the described production method, relatively low concentrations of ethyl alcohol are used during DNA precipitation. To do this, ethyl alcohol is added to the cooled DNA solution, pre-cooled to 0-10 ° C, to a concentration of 37-38 vol.%, Followed by stirring the resulting solution. Water-soluble impurities still remain dissolved under these conditions. The precipitated unfragmented high molecular weight DNA preparation was separated by filtration and washed with ethyl alcohol.

The next task is to obtain the sodium salt of fragmented heterogeneous DNA of a given size. As already mentioned, ultrasonic treatment of the DNA solution is usually used for this, which is a rather slow and inconvenient process. In the present invention, for the fragmentation of DNA, the process of passage of a high molecular weight DNA solution through a narrow gap at a high pressure drop, for example, through a gap of a high pressure homogenizer, is used. This procedure has significantly accelerated the process. In addition, the degree of fragmentation can be changed by changing the pressure drop across the slit and the number of solutions passing through the slit (Tables 1 and 2). For this, DNA is dissolved in an aqueous solution of NaCl at a concentration of 0.01-4.5 M at a temperature not exceeding 50 ° C. The resulting solution is passed through a high-pressure homogenizer at a pressure of up to 2000 kg / cm ² , the resulting fragmented DNA is treated with alcohol to obtain a concentration of 40 to 90%, the sodium salt of the fragmented DNA precipitated in alcohol is separated by centrifugation, after which the precipitate is washed with 96% alcohol and dried in drying oven at a temperature not exceeding 50 ° C to obtain fragmented heterogeneous DNA powder of sodium salt.

During the study, the following results were obtained:

Table 1 Dependence of the characteristic viscosity of DNA fragments and its average molecular weight on the excess pressure after four passes of its solution through the slit of a high-pressure homogenizer. Overpressure, atm. [η] · 10 ^-2 , cm ³ / g Molecular weight, kDa 125 8.4 ~ 1000 225 7.2 ~ 800 500 5.0 ~ 600 900 3.6 ~ 450 1200 1.7 ~ 300

Table 2. Dependence of the intrinsic viscosity of DNA fragments and its average molecular weight on the number of passes of its solution through the slot of a high-pressure homogenizer at an overpressure of 900 atm. The number of passes [η] · 10 ^-2 , cm ³ / g Molecular weight, kDa one 4.8 ~ 600 2 4.3 ~ 550 3 3.9 ~ 500 four 3.6 ~ 450

where [η] is the intrinsic viscosity of the DNA preparation.

Values of intrinsic viscosity were estimated by linear extrapolation of the dependence of the reduced viscosity of the DNA solution to zero DNA concentrations. The reduced specific viscosity was measured on an automatic rotational viscometer manufactured by SKB Biological Instrumentation RAS. At least four points with DNA concentrations in the range from 0.1 to 3.5 mg / ml were measured for each line. The initial average molecular weight of DNA molecules was at least 7000 kDa.

Thus, the achievement of the task is achieved - the simplification of the method of obtaining sodium salt of DNA while maintaining its characteristics.

A specific example of the implementation of the method of obtaining the sodium salt of heterogeneous deoxyribonucleic acid (DNA) from the testes of animals and their sperm:

1. To obtain DNA, animal raw materials are taken: fish milk, cattle seed, sperm.

2. Raw materials, such as fish milk or animal testes (0.5 kg), are ground in a meat grinder or any other homogenizer.

3. The crushed mixture is transferred to a container with a stirrer and heating, where 20 l of a 1-3% solution of sodium dodecyl sulfate (SDS), preheated to 35-60 ° C (temperature depends on the type of raw material) is added. The mixture is constantly stirred at an arbitrary speed for 2 hours while maintaining the above temperature.

4. After 2 hours, the coagulant — permanganate of an alkali or alkaline earth metal (in particular, KMnO ₄ ), is added to the solution in dry form (or in the form of a concentrated solution). The amount of coagulant is calculated from the need to obtain 0.0005 M - 0.03 M solution (depending on the type of raw material). For milk of fish 1-10 mm. The whole mixture at a temperature of 35-60 ° C is mixed for 30 minutes (the temperature depends on the type of raw material).

5. After this, the ionic strength of the solution rises to 1.0-3.0 M by adding dry (or in the form of a concentrated solution) sodium chloride to the solution (other salts can also be used). The temperature rises to 62 ° C and the mixture is stirred for another 1.0-2.0 hours.

6. After this, a precipitate is formed which contains only impurities. The precipitate is separated by filtration on a filter with 10 μm pores (ceramic, paper, etc.).

7. After filtering, a clear, viscous, unpainted solution is obtained that contains high molecular weight (VM) DNA and a small amount of water-soluble impurities.

8. The solution of VM DNA is cooled to 0-20 ° C and pre-chilled alcohol is added to a concentration of 37 vol% (for ethyl). In the case of other alcohols, the lowest possible concentration is added, leading to the loss of DNA from the solution.

9. The sodium salt of VM DNA is precipitated in the form of fibers that separate well from the solution. The planted product is separated by filtration (or other mechanical methods, for example, by winding on glass rods), washed with alcohol and stored under 96% ethyl alcohol.

Obtaining fragmented (low molecular weight) sodium salt of DNA from unfragmented (high molecular weight) DNA:

1. Obtaining fragmented DNA from VM DNA of any origin consists in the fact that VM DNA is dissolved in a 0.01-4.5 M aqueous solution of sodium chloride at a temperature not exceeding 50 ° C.

2. Next, the solution is passed through the hole in the device that creates excess pressure, for example, this device may be a high pressure homogenizer.

3. Single or multiple passage of the solution through a slit-shaped or other narrow hole at elevated overpressure from 1 to 2000 atm allows to obtain DNA fragments of a given size (from 150 to 1500 kDa).

4. The solution with NM DNA is treated with alcohol (for example, ethyl) with the creation of an alcohol concentration of 30 to 90%. The sodium salt of NM DNA is precipitated, which is separated by centrifugation.

5. The separated sodium salt product NM DNA is dried at a temperature not exceeding 50 ° C.

Comparative characteristics of DNA preparations from sturgeon milk obtained using various methods are presented in table 3.

Table 3, confirming the increase in the yield of the final product by the claimed method Comparative characteristics of DNA preparations from sturgeon milk obtained using various methods. Options/
Method Exit The disadvantages of the method compared to the proposed one. The content of impurity proteins (%) Average molecular weight (kDa) Hyperchromic effect (%) This application 5.5% ~ 0.5% ~ 7000> M> ~ 300 (optional, depending on the pressure used) -40% Patent RU No. 20055724 4.3% Slow filtration of the extract. Unproductive ultrasonic treatment. Slow concentration by ultrafiltration. The need to wash the target product by diafiltration. ~ 1.5% 400 -40% Patent RU No. 2017496, A 61 K 35/60, C 07 21/04 12/28/1990 ~ 1.5% 450 Note: All isolations were carried out according to the methods described in the patents using milk belonging to one batch. The amount of DNA was determined spectrophotometrically. The protein content was determined by the Lowry method. The magnitude of the hyperchromic effect was estimated by the change in optical density as a result of acid
DNA denaturation.

This method of obtaining the sodium salt of heterogeneous deoxyribonucleic acid (DNA) from the testes of animals and / or their sperm can be used in the pharmaceutical and cosmetic industries to obtain modern medicines and cosmetics, as well as in the food industry for food additives.

Claims (1)

The method of obtaining the sodium salt of heterogeneous deoxyribonucleic acid from the testes of animals and / or their semen, including grinding and homogenizing the feedstock in a detergent solution, processing the solution at an elevated temperature while stirring the solution, separating the liquid phase by filtration and precipitating the product with alcohol, characterized in that the homogenization carried out in a 1-4% solution of sodium dodecyl sulfate in distilled water at a temperature of 35-60 ° C and stirring the solution for 1.5-4 hours, after what is added to the solution a coagulant - permanganate of an alkaline or alkaline earth metal to obtain a concentration of coagulant in a solution of 0.0005 - 0.03 M, the resulting solution is stirred at a temperature of 35-60 ° C for 25-90 minutes, after which the ionic strength of the solution is increased to 1-3 M, and the solution is heated to 60-62 ° C and the solution is stirred for 1-3 hours at the above temperature, then the precipitate of impurities obtained during coagulation is separated by filtration through a filter with pores from 1 to 25 μm in size, the filtered solution of unfragmented high molecules The DNA is cooled to a temperature of 0-20 ° C, then alcohol is added to the solution to a final concentration of 27-43.5 vol.%, and then the resulting solution is stirred and the resulting alcohol-precipitated preparation is separated by filtration, washed with 96% ethanol and stored under ethanol, the resulting DNA preparation is then dissolved in an aqueous NaCl solution of a concentration of 0.01-4.5 M at a temperature not exceeding 50 ° C, the resulting solution is passed through a high pressure homogenizer at a pressure of up to 2000 kg / cm ² , processed obtained fragmented DNA annuyu alcohol to give a concentration of 40 to 90%, the precipitated sodium in alcohol fragmented DNA is separated by centrifugation, whereupon separated precipitate was again washed with alcohol and dried in an oven at a temperature not higher than 50 ° C to give a powder.