Method for production of plasmid dna from bacterial cells

FIELD: medicine.

SUBSTANCE: method includes alkaline lysis of bacterial cells and depositing of cellular walls by centrifugation. Afterwards supernatant is filtered, and RNA admixture is removed from produced filtrate by means of filtrate treatment with RNA-ase. Then plasmid DNA is deposited with isopropyl alcohol. Then divisional fractioning of plasmid DNA is carried out with ethyl alcohol, when first ethyl alcohol is added to solution of plasmid DNA to concentration of 45-53%, then ethyl alcohol is added to concentration of 55-58%, and in the end ethyl alcohol is added to concentration of 60-67%. Afterwards lipopolysaccharide remains are removed with the help of reverse-phase sorbent with hydrophobised surface.

EFFECT: improved cleanliness and yield of plasmid DNA.

3 cl, 1 tbl, 4 ex

 

The invention relates to biotechnology and genetic engineering, namely to technology developments and allocation of preparative quantities of pyrogen-free DNA, and can be used in medicine and the pharmaceutical industry.

In the last decade gynomonoecy gained recognition as a method that allows to obtain a stable immune response against the desired antigen. DNA vaccines are currently in use not only for the immunization of animals; intensively developed DNA vaccines intended for the induction of humoral and cellular response in humans [1, 2]. Such vaccines are based, generally, on the use of plasmid DNA, and, although they are less effective than vaccines based on viral vectors, their application is approved by the who, because it has no obvious long-term effects related to the integration of viral DNA into the genome. To increase the efficiency of transfection of cells plasmid DNA using special delivers design, but to obtain a positive effect of immunization is necessary to use relatively high doses of DNA. This fact requires the use for immunization "pure" DNA, which is DNA that is free from impurities that reduce the efficiency of transfection (such as bacterial DNA and RNA), as well as immunogenic (such as bacterial proteins, proteoglycans, glycoprotein the us etc) and toxic impurities (such as bacterial lipopolysaccharide (LPS)).

One of the requirements for pharmaceuticals DNA, is the absence of endotoxins (in particular, bacterial LPS). The methods used to allocate pyrogen-free DNA can be divided into three types: based on the specific precipitation of DNA from solutions based on the extraction of LPS from the purified DNA solution, and the specific binding of immobilized ligands with LPS.

A method of obtaining plasmid DNA, based on the selective extraction of LPS from the DNA solution [3]. The method consists in the fact that the solution of plasmid DNA obtained by the method of alkaline lysis, add Triton X-114 to 1%, stirring, cooled in an ice bath until the solution becomes homogeneous. Then the resulting solution was heated to 25°C and separated into two phases by centrifugation (38000 g, 37°C, 30 min) and subsequent incubation at 37°C for 30 minutes Three times repeating this extraction allows to remove up to 99% of LPS (up to 64 EE/mg). Subsequent uninominal chromatography reducing the concentration of LPS to 2.5 EE/mg plasmid DNA, as well as to remove residual Triton X-114, RNA and proteins.

The disadvantages of this method are the difficulty of removing residual Triton from the DNA solution and the high loss of plasmid DNA in the extraction step LPS Triton X-114, which is 50-60%.

A method of obtaining plasmid DNA by removing impurities bacterial LPS from DNA solutions using ion-exchange chromatography [5]. The essence of the method consists in using a column filled with cellulose beads with immobilized on the surface of polyethylenimine or diethylaminoethyl. These adsorbents have a high ability to bind endotoxins.

The disadvantages of this method include only a single possibility of using the column is, the low efficiency of removal of impurities of endotoxin from solution and high loss of DNA.

There is a method of obtaining and purification of plasmid DNA, comprising three phase chromatography [6]. The original bacterial lysate was applied to a column of Sepharose 6 Fast Flow (Amersham Biosciences), then the collected eluate was applied to a column of Sepharose 6 Fast Flow, in which the sorbent contained immobilized sulfur-containing aromatic residues (2-mercaptopyridine, 2-pyridine ethandiol). Two-phase chromatography is possible to reduce the content of endotoxin in the resulting eluate 319 times compared with the original lysate (according to LAL-test 470 and 150,000 EE/mg plasmid, respectively). The final stage of this method is to clean the obtained eluate column on SOURCE 30Q (Amersham Biosciences), which allows to reduce the amount of endotoxin to 9 EE/mg of plasmid DNA.

The disadvantages of this method are the duration, complexity, resursozatratno because of the high cost and disposable chromatographic sorbents and receiving small amounts of purified plasmid DNA.

A method of obtaining plasmid DNA pharmaceutical quality [7], including the production of cells containing plasmid DNA, obtaining a lysate containing plasmid DNA, by the destruction of cells by the method of continuous alkaline lysis, concentration of the lysate by OS the input voltage to a suitable agent, anion-exchange chromatography, chromatography with the formation of the triple helix, hydrophobic chromatography and the final stage of diafiltration and/or buffer exchange. At this stage of diafiltration includes the following stages: collection solution with the latest chromatographic stage; the implementation of the first stage of diafiltration against buffer Tris/NaCl; implementation of the second phase of diafiltration against salt solution under conditions suitable to control the final concentration of the buffer to stabilize the pH of the final preparation of plasmid DNA.

The disadvantages of this method are the duration and intensity, as well as large losses of the target product at multiple stages chromatography.

There is a method in which to remove impurities of endotoxin from the preparation of plasmid DNA using commercially available media, in particular agarose particles with immobilized polymyxin B (Pierce Detoxi-Gel beads [8]. Polymyxin b is a cyclic peptide produced by Bacillus polymyxa, and capable with high affinity to bind LPS. This method includes the following stages: isolation of plasmid DNA from E.coli cells by any standard method; the precipitation of plasmid DNA in 96% ethanol, followed by dilution of DNA in pyrogen-free TE Buffer (10 mm Tris-HCl, 1 mm EDTA, pH 8.0) to a concentration of 1 mg/ml; preparation of 50% WM is ansii agarose particles with immobilized polymyxin B; chromotagraphy with a mixing ratio of 2:1:1 (suspension of agarose particles with polymyxin/Buffer-TNE (10 mm Tris-HCl, pH 8.0, 0.25 M NaCl, 1 mm EDTA) / plank in TE Buffer).

The disadvantages of this method include the difficulty of regeneration of used speakers and a high content of LPS in a purified preparation of plasmid DNA (from 10 to 50 EE/mg DNA). In addition, since polymyxin In activates blood stimulation nephrotoxin and neurotoxin, mixture of polymyxin b in the preparation of plasmid DNA, intended for introduction into the human, is invalid.

The most closest to the claimed method of the prototype, is a method for plasmid DNA for pharmaceutical quality, including selective precipitation of plasmid DNA from bacterial lysate using the cationic detergent is BECOMING (cetyltrimethylammonium bromide, CH3(CH2)15N(CH3)3Br), followed by purification of the obtained plasmid DNA using anion-exchange chromatography [9]. In this way, the first step is an alkaline lysis of bacterial cells, which carry out the removal of impurities RNA by addition of RNase a to a concentration of 100 μg/ml of the resulting lysate purified from cell walls by centrifugation followed by filtration through 22-25 µm filter. The second step is the selective deposition plasmids the th DNA by adding to the filtrate BECOMING to a final concentration of 2 g/L. The precipitated DNA was dissolved in buffer (0.60 M NaCl, 25 mM Tris-HCl, mM EDTA, pH 7.4) over night. Additional cleaning preparations of plasmid DNA is administered by the anion exchange chromatography using weak anion-exchange membrane cartridges (MA 75 D, Sartorius). In the last step, the fractions containing DNA are pooled and transferred to a physiological solution by gel filtration (Sephadex G-25). This method allows you to get the preparations of plasmid DNA containing up to 5 EE of endotoxin on milligrams of plasmid DNA.

The disadvantage of this method is that it leads only to a partial purification of DNA (concentration of protein in an average of 100 μg/mg of plasmid DNA). In addition, loss of plasmid DNA only at the stage of deposition of BECOMING th is not less than 50%, and the use of ion-exchange chromatography increases the complexity and resource intensity of way.

An object of the invention is to increase the purity and yield of the target product.

The goal of the project is achieved by the claimed method consists in the following.

The biomass of bacterial cells obtained by culturing the producers of recombinant plasmids are lysed using alkaline lysis. The resulting lysate purified from cell walls by centrifugation followed by filtration through a folded Phil is Tr (2 layers of filter paper or Whatman 3MM).

To remove impurities RNA to the filtrate add a solution of RNA-basics And up to a concentration of 50 μg/ml and incubated in a water bath at room temperature for 1 h To remove the enzyme to the solution add 1/600 of the volume of 10% SDS, 1/1000 volume of β-mercaptoethanol, mix, add 1/60 volume of 3 M potassium acetate, pH 5.5, and incubated in ice for 30 minutes followed by centrifugation at 24000 g for 25 minutes at 4°C. the resulting supernatant is filtered through a folded paper filter.

Plasmid DNA is precipitated from the resulting filtrate by adding 0.7 volume of isopropanol followed by centrifugation. The precipitated DNA was washed with 70% ethanol and dissolved in Buffer 1 (25 mm Tris-HCl, 20 mm EDTA, pH 8.0; the rate of 1 ml buffer per 1 g initial biomass).

Lipopolysaccharide and DNA differ in solubility, therefore, the preparation of plasmid DNA, resulting from deposition by ispropanol, fractionary sequential precipitation with ethyl alcohol. To do this, to the solution of plasmid DNA add ethyl alcohol to a concentration of 45-53%, followed by incubation in ice for 30 minutes After centrifugation at 27000 g for 20 minutes at 4°C. take the supernatant and add 3 M ammonium acetate to a concentration of 60 mm and in ethanol to a concentration of 55-58%, followed by incubation in ice for 15 minutes After zentrifugenbau 27000 g for 20 minutes at 4°C. take the supernatant and add ethyl alcohol to a concentration of 60-67%, followed by incubation at room temperature for 30 minutes After centrifugation 27000 g for 20 minutes at room temperature, the supernatant removed, the residue dissolved in 3-5 ml of Buffer 1.

For a deep cleaning of the preparation of plasmid DNA from impurities LPS using a suspension of commercially available reversed-phase sorbent with gidrofobizirovannogo surface, mostly phenyl-sepharose/octyl-agarose/butyl-agarose, in an amount necessary to clear one sample of plasmid DNA (25-35 ál perseverate/octyl-agarose/butyl-agarose per 100 PU (AR260) plasmid DNA). The suspension is washed 3 times 5-fold excess of pyrogen-free water by centrifugation at 17000 g. To the precipitate phenyl-sepharose/octyl-agarose add plasmid DNA and incubated at room temperature on a rotary drum-type mixer (rotational speed 10 rpm) for 4 hours. Centrifuged at 17000g for 10 minutes at room temperature. The supernatant (solution of plasmid DNA) is transferred into a new tube. Plasmid DNA is filtered under sterile conditions through a membrane filter with a pore diameter of 0.22 micron.

The defining difference of the proposed method from the prototype are:

1) Preparation of plasmid DNA, obtained by precipitation with isopropanol, fractional fractionary serial three-fold by precipitation with ethyl alcohol, taken in optimalnoe increasing concentration from 45 to 67%, freeing preparation of plasmid DNA from impurities LPS and proteins.

2) After fractionation with alcohol preparation of plasmid DNA additionally purified using reversed-phase sorbent with gidrofobizirovannogo surface, mostly phenyl-sepharose, octyl-agarose or butyl-agarose, taken in an amount of 25-35 μl of suspension per 100 PU (AR260) plasmid DNA, which allows for deeper cleaning of the preparation of plasmid DNA from impurities LPS.

The invention is illustrated by the following examples of specific performance.

Example 1. Isolation of plasmid DNA from biomass, fraction fractionation plank ethyl alcohol and purified using octyl-agarose.

Cells of E. coli DH5αF', transformed with recombinant plasmid pcDNA-TCI, increased to OD600=1.5 in the fermenter LKB. Cell suspension was centrifuged at 5500 g for 10 minutes at 4°C, supernatant was removed and cells resuspendable in pre-cooled in ice Buffer 1 (25 mm Tris-HCl, 20 mm EDTA, pH 8.0) at a rate of 200 ml buffer 25 g of cells. Cell suspension was again centrifuged at 5500 g for 10 minutes at 4°C, the precipitated cells resuspendable in pre-cooled Buffer 1 (25 g cells, the total volume of the suspension should be 200 ml) and incubated in ice for an orbital shaker for 10 min (150 rpm, Ampl is there 5). Then the cells were added 240 ml of Buffer 2 (1% SDS, 0.2 M NaOH) and incubated in ice for orbital rocking no more than 5 minutes (150 rpm, amplitude 5). Next was added 240 ml of Buffer 3 (3 M potassium acetate, pH 5.5), then 45 ml of a saturated solution of potassium perchlorate, and incubated in ice for an orbital shaker for 15 min (150 rpm, the amplitude of 5), followed by centrifugation at 24000 g for 25 minutes at 4°C.

The supernatant was filtered through a folded filter paper (2 layers of filter paper or Whatman 3MM).

To the filtrate was added a solution of RNA-basics And up to a concentration of 50 μg/ml and incubated in a water bath at room temperature for 1 h After incubation the solution was added 1/600 of the volume of 10% SDS, 1/1000 volume of β-mercaptoethanol were mixed, was added 1/60 of the amount of Buffer 3, and incubated in ice for 30 minutes followed by centrifugation at 24000 g for 25 minutes at 4°C. the Supernatant was filtered through a folded filter paper (2 layers of filter paper).

To the filtrate was added 0.7 volume of isopropanol (to a final concentration of 40%), mixed and incubated at room temperature for 10 minutes Plasmid DNA precipitated with centrifugation at 24000 g for 20 min at 4°C. the Supernatant decantation, the precipitate DNA was added to 50 ml of 70% ethanol and stirred. Drove volume of ethanol, 200 ml, was centrifuged at 24000 g for 10 m is the chick at 4°C and carefully remove supernatant. The precipitate was dissolved in Buffer 1 (1 ml buffer per 1 g initial biomass)to the precipitate DNA was added 0.7 volume of the required volume of buffer and left overnight on an orbital shaker in ice (150 rpm, amplitude 5). Then brought the volume of solution to volume, numerically equal to the weight of the original biomass.

Then there was the fractional separation of plasmid DNA with ethanol. To do this, to the solution of plasmid DNA was added 1.2 volume of 96% ethanol (to final concentration of 52.4%) and incubated on ice for 30 min followed by centrifugation at 27000 g for 20 minutes at 4°C. the Supernatant was transferred into new tubes, was added 0.02 volume of 3 M ammonium acetate and 0.14 volume of 96% ethanol (to a final concentration 56.7%) and incubated on ice for 15 minutes

Was centrifuged at 27000 g for 20 minutes at 4°C. the Supernatant was transferred into new tubes, was added to the supernatant 0.11 volume of 96% (to a final concentration of 60.7%ethanol, incubated for 30 min at room temperature and was centrifuged at 27000 g for 20 minutes at room temperature. After centrifugation the supernatant was removed, the residue was dissolved in 3 ml of Buffer 1. The DNA concentration was measured spectrophotometrically.

Then there was the cleaning plank using octyl-agarose. For this purpose a suspension of octyl-agarose in to the Icesave, necessary to clean one sample of plasmid DNA (35 ál of octyl-agarose per 100 PU (AR260) plasmid DNA), washed 3 times 5-fold excess of pyrogen-free water (double-distilled water tested for purity endotoxin) by centrifugation at 17000 g. To the precipitate activegamez added plasmid DNA and incubated at room temperature on a rotary drum-type mixer (rotational speed 10 rpm) for 4 hours, followed by centrifugation at 17000 g for 10 minutes at room temperature. The supernatant (solution of plasmid DNA) was filtered under sterile conditions through a membrane filter with a pore diameter of 0.22 micron.

In the target preparation of plasmid DNA, and obtained in the fractional deposition of sediments, dissolved in Buffer 1, was determined by the concentration of plasmid and genomic DNA, proteins and LPS. To determine the impurity content of protein in the preparations of plasmid DNA was used fluorescent method based on the use of specific fluorescent dye to protein - NanoOrange Protein Quantitation Kit (Molecular Probe, USA). The measurement of the concentration of plasmid DNA and evaluation of impurities in the genomic DNA of E. coli was performed using quantitative real-time PCR. For determination of endotoxin in samples of plasmid DNA was used the AL-test (associates of Cape Cod Incorporated, USA).

Indicators of the quality of the target product are presented in the table.

Table
StageThe number of pcDNA-TCI obtained from 25 g of E. coli cells according to quantitative PCR mgThe content of the genomic DNA mgThe content of FSC EEThe protein content, ág
The solution of plasmid DNA after precipitation with isopropanol194≥125000-
Precipitate DNA after precipitation 52.4% ethanol0.953.6≥112500-
Precipitate DNA after precipitation 56.7% ethanol1.80.46--
Precipitate DNA after precipitation 60.7% ethanol16.20.00013≤625<160
The solution of plasmid DNA after purification PR is using octyl-agarose 12.50.0001<62.5<120

From the data obtained it follows that the sequential deposition of a solution of plasmid DNA and 52.4 56.7% ethanol leads to the almost complete removal of impurities genomic DNA and proteins and the removal of more than 99% of the IDPs. Purification of plasmid DNA using octyl-agarose significantly reduce impurities LPS and 1 mg of plasmid DNA does not contain more than 5 EE LPS, not more than 10 nanograms of genomic DNA and less than 10 micrograms of proteins. Loss of plasmid DNA after alcohol fractionation and purification using octyl-agarose was approximately 35%.

Example 2. Isolation of plasmid DNA from biomass, fraction fractionation plank ethyl alcohol and purified using a phenyl-sepharose.

Plasmid DNA was isolated from cells of E. coli DH5αF' analogously to example 1 to stage fractionation ethanol.

Then to the solution of plasmid DNA was added 0.882 volume of 96% ethanol (to final concentration of 45%) and incubated on ice for 30 min followed by centrifugation at 27000 g for 20 minutes at 4°C. the Supernatant was transferred into new tubes, was added 0.02 volume of 3 M ammonium acetate and 0.272 volume of 96% ethanol (to final concentration of 55%) and incubated on ice for 15 minutes

what was centrifugally at 27000 g for 20 minutes at 4°C. The supernatant was transferred into new tubes, was added to the supernatant 0.14 volume of 96% ethanol (to final concentration of 60%), incubated for 30 min at room temperature and was centrifuged at 27000 g for 20 minutes at room temperature. After centrifugation the supernatant was removed, the residue was dissolved in 4 ml of Buffer 1. The DNA concentration was measured spectrophotometrically.

Then there was further purification of plasmid DNA as in example 1, except that the adsorbent used phenyl-sepharose and added it at the rate of 30 μl of the suspension phenyl-sepharose 100 PU (AR260) plasmid DNA.

After cleaning plank using phenyl-sepharose, 1 mg of plasmid DNA contained less than 10 nanograms of genomic DNA, 2.5 EE FSC and less than 10 micrograms of proteins. Loss of plasmid DNA after fractionation with alcohol and clean with phenyl-sepharose was approximately 30%.

Example 3. Isolation of plasmid DNA from biomass, fraction fractionation plank ethyl alcohol and purified using a phenyl-sepharose.

Plasmid DNA was isolated from cells of E. coli DH5αF' analogously to example 1 to stage fractionation ethanol.

Then to the solution of plasmid DNA was added 1.233 volume of 96% ethanol (to final concentration of 53%) and incubated on ice for 30 min is followed by centrifugation at 27000 g for 20 minutes at 4°C. The supernatant was transferred into new tubes, was added 0.02 volume of 3 M ammonium acetate and 0.162 volume of 96% ethanol (to final concentration of 58%), and incubated on ice for 15 minutes

Was centrifuged at 27000 g for 20 minutes at 4°C. the Supernatant was transferred into new tubes, was added to the supernatant 0.31 volume of 96% ethanol (to a final concentration of 67%), incubated for 30 min at room temperature and was centrifuged at 27000 g for 20 minutes at room temperature. After centrifugation the supernatant was removed, the residue was dissolved in 5 ml of Buffer 1. The DNA concentration was measured spectrophotometrically.

Then there was further purification of plasmid DNA as in example 1, except that the adsorbent used phenyl-sepharose and added it at the rate of 25 μl of the suspension phenyl-sepharose 100 PU (AR260) plasmid DNA.

After purification of plasmid DNA using phenyl-sepharose, 1 mg of plasmid DNA contained less than 10 nanograms of genomic DNA, less than 5 EE FSC and less than 10 micrograms of proteins. Loss of plasmid DNA after fractionation with alcohol and clean with phenyl-sepharose was approximately 30%.

Example 4. Isolation of plasmid DNA from biomass, fraction fractionation plank ethyl alcohol and purified using a butyl-agarose.

Plasmid is the TC was extracted from cells of E. coli DH5αF' analogously to example 1 to stage fractionation ethanol.

Then to the solution of plasmid DNA was added 1.1 volume of 96% ethanol (to final concentration of 50.3%) and incubated on ice for 30 min followed by centrifugation at 27000g for 20 minutes at 4°C. the Supernatant was transferred into new tubes, was added 0.02 volume of 3 M ammonium acetate and 0.17 volume of 96% ethanol (to a final concentration of 56%) and incubated on ice for 15 minutes

Was centrifuged at 27000 g for 20 minutes at 4°C. the Supernatant was transferred into new tubes, was added to the supernatant 0.3 volume of 96% ethanol (to final concentration of 65.2%), incubated for 30 min at room temperature and was centrifuged at 27000 g for 20 minutes at room temperature. After centrifugation the supernatant was removed, the residue was dissolved in 4 ml of Buffer 1. The DNA concentration was measured spectrophotometrically.

Then there was further purification of plasmid DNA as in example 1, except that the adsorbent used butyl-agarose was added at the rate of 30 μl of the suspension butyl-agarose per 100 PU (AR260) plasmid DNA.

After cleaning plank using butyl-agarose, 1 mg of plasmid DNA contained less than 10 nanograms of genomic DNA, 2.5 EE FSC and less than 10 micrograms of proteins. Loss of plasmid DNA after fractionation with alcohol and clean if help is butyl-agarose was approximately 30%.

The method allows to obtain a pyrogen-free plasmid DNA in preparative quantities with high output.

Sources of information

1. Donnelly, J.J, Ulmer, J.B., Shiver, J.W., Liu, M.A., DNA vaccines. Ann. Rev. Immunol. - 1997. - V.15. P.617-648.

2. Gurunathan, S., Klinman, D.M., Seder, R.A., DNA vaccines: immunology, application, and optimization. Ann. Rev. Immunol. - 2000. - V.18. P.927-974.

3. Rozkov, A., Larsson, B., Gillström, S., Björnestedt, R., Schmidt, S.R., Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol Bioeng. 2008. - V.99. - P.557-566.

4. Yasuda, K., Kawano, H., Yamane, I., et all, Restricted cytokine production from mouse peritoneal macrophages in culture in spite of extensive uptake of plasmid DNA. Immunology. - 2004. - V.111. - P.282-290.

5. Mitzner, S., Schneidewind, J., Falkenhagen, D., Loth, F., Klinkmann, H., Extracorporeal endotoxin removal by immobilized polyethylenimine. Artif. Organs. 1993. - V.17. - P.775-781.

6. Lemmens, R., Olsson, U., Nyhammar, T., Stadler, J., Supercoiled plasmid DNA: selective purification by thiophilic/aromatic adsorption. J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 2003. - V.784. - P.291-300.

7. Application RU # 200600579 "Method of production of plasmid DNA for pharmaceutical quality, With 12 N 15/10, on. 27.10.2006.

8. Montbriand, P.M., Malone, R.W., Improved method for the removal of endotoxin from DNA. J. Biotechnol. 1996. - V.44. - P.43-46.

9. Syren P.O., Rozkov, A., S.R. Schmidt, P. Stromberg, Milligram scale parallel purification of plasmid DNA using anion-exchange membrane capsules and a multi-channel peristaltic pump. J. of Chromatography. 2007. - V.856. - P.68-74.

1. The way to obtain plasmid DNA from bacterial cells, including alkaline lysis last, the deposition of cell walls by centrifugation, filtration of the supernatant, removing from the resulting filtrate impurities RNA PU who eat processed filtrate RNA Asai, the precipitation and purification of plasmid DNA, characterized in that the deposition of plasmid DNA spend isopropyl alcohol, and then carry out a fractional distillation of plasmid DNA with ethanol, to the solution of plasmid DNA first, add ethyl alcohol to a concentration of 45-53%, then add ethyl alcohol to a concentration of 55-58% and at the end add ethyl alcohol to a concentration of 60-67%, followed by removal of residual LPS using reversed-phase sorbent with gidrofobizirovannogo surface.

2. The method according to claim 1, characterized in that the deposition of plasmid DNA using isopropyl alcohol to a final concentration of 40%.

3. The method according to claim 1, characterized in that the sorbent used octyl-agarose based 35 μl of suspension per 100 PU (AR260) plasmid DNA, phenyl-sepharose based 25-30 μl of suspension per 100 PU (AR260) plasmid DNA or butyl-agarose for 30 µl of the suspension per 100 PU (AR260) plasmid DNA.



 

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2 cl, 3 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, in particular to extraction of physiologically active compounds and can be used in medicine. Dry yeast is suspended in a 1-2% aqueous solution of oleic acid titrated with an alkali to pH 7-8. The suspension is kept at 99-102°C for 40-60 minutes. Lysate cooled to 35-45°C is centrifuged without cooling. The supernatant fluid is discharged and NaCl is added to the discharged fluid until achieving concentration of 2-3 M. The suspension is kept for 20-24 hours at room temperature and centrifuged without cooling. Low-polymeric RNA is extracted from the obtained supernatant and the precipitate-extraction cake is washed with two portions of 2-3 M solution of NaCl and with four portions of 92-97% ethanol via successive resuspension at room temperature and centrifuging. High-polymeric RNA is extracted from the obtained extraction cake using distilled water and then dried using a conventional method. Ethanol is reclaimed through distillation. Natural oligoribonucleotides are extracted from the low-polymeric RNA through gel filtration on Sepharose-4B.

EFFECT: invention enables to obtain high-polymeric RNA with considerably high molecular weight as a result of using dry baker's yeast without preliminary soaking or using high temperature.

5 cl, 1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: sample is subjected to lysis in buffer on the basis of anionic detergent (SDS), lysate is subjected to purification and deoxyribonucleic acid is precipitated with ethanol or isopropanol. Lysate purification is performed by means of detonation nanodiamond, selectively sorbing mixtures of non-nuclear nature. Detonation nanodiamond is preliminarily burnt at temperature interval 400-700°C in vacuum 10-2-10-3 Torr. Obtained DNA solution contains both short and long fragments. Claimed method of isolation and purification of DNA allows to avoid application of such highly toxic compounds as phenol or chloroform. Contamination of obtained DNA with chemically active substances is prevented.

EFFECT: in case of accidental contact of detonation nanodiamond with final solution, polymerase chain reaction is not blocked, it is possible to separate non-soluble of detonation nanodiamond sediment from DNA solution.

3 dwg, 1 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to methods of primer-dependent propagation of nucleic acids in immobilised media, particularly to methods of detecting products of such propagation. The invention discloses an improved method of identifying changes in a nucleotide sequence and determining the number of molecules of nucleic acids as separate molecules and contained in cells or in tissue fragments. The invention enables detection of molecular colonies of nucleic acids through fluorescence of at least growth of colonies in an immobilised medium (in real time) without an additional hybridisation step and without unsealing the chamber containing the medium. If the colony is formed by more than one target molecule, the invention enables to determine the number of target molecules from which the given colony originates. This in turn enables determination of spatial location and local concentration of RNA or DNA molecules of the given type in cells or in tissue fragments contained in the gel at the polymerisation step.

EFFECT: invention can be widely used as a diagnosis method and scientific research instrument.

55 cl, 11 dwg, 12 ex

FIELD: medicine.

SUBSTANCE: method facilitates linkage of sequences, coding immunoglobulin variable regions, T-cells receptors or B-cells receptors. Method is instrument of higher effectivity for making sequence data libraries. Capability of multiple RT-PCR with chain extension by interruption with employment of matrix, derived from single cell, provides highly effective creation of sister pairs libraries.

EFFECT: method is effective for linkage of two or few nucleotide sequences, coding domens or subunits of heteromeric protein as a result of single reaction performance.

51 cl, 25 dwg, 27 tbl, 14 ex

FIELD: agriculture.

SUBSTANCE: at stage of vegetable samples' homogenisation hydroxy derived from benzoic acid is added to a lytic buffer in amount of 20-30 mg for a sample. The lytic buffer contains 4 mg guanidine hydrochloride, 0.2 M sodium acetate, 25 mM EDTA , 2.5% kollidon, 20% sarcosyl.

EFFECT: efficiency upgrading RNA's educing, more successful PCR's process and revealing of viruses in vegetable sphere.

2 dwg, 2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: one version of the proposed method involves passing sugar-containing liquid, particularly diffusion juice, together with a gas stream through a closed aeration chamber, addition of lime to the aerated sugar-containing liquid and then passing carbon dioxide. According to another version of the method, sugar-containing liquid is passed through a closed vacuum chamber and then through a closed aeration chamber, lime is added to the treated sugar-containing liquid, after which carbon dioxide is passed through the sugar-containing liquid.

EFFECT: reduced chromaticity of sugar-containing liquid.

5 cl, 4 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: replaceable microfluid module for automated recovery and purification of nucleic acids from biological samples contains reagent reservoirs and reaction capacities, microchannels and valves required to handle the liquids in the module. The module comprises an inleak chamber for introduction of the biological sample, a lysis reservoir containing a membrane, reservoirs for dissolved lytic buffers, a microcolumn containing a solid-phase sorbent for binding nucleic acids, a reservoir for flush buffer and ethanol to discharge residual proteins and salt crystals from the microcolumn, an eluting buffer reservoir, an outlet port compatible to a standard microtube, a waste collection reservoir. The method for recovery and purification of nucleic acids from biological samples involves filtration of a sample through the membrane, lysis of the microorganism cells kept on the membrane, binding of nucleic acids on the microcolumn, washing nucleic acids from proteins and salt crystals, elution of nucleic acids from the microcolumn in the tube. All the stages of recovery and purification are carried out in automatic mode in the microfluid module that isolates the sample from environment.

EFFECT: invention allows reducing cost price of the nucleic acid preparation.

14 cl, 17 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: cardiac hystiocytes are proliferated by inducing of cyclin and CDK expression in cardiac hystiocytes and by inhibiting function or activity of protein of Cip/Kip family or by inhibiting production of protein of Cip/Kip family. There is described expression vector that contains a cyclin gene, a cyclin-dependent kinase gene and one or several agents chosen from the group consisting of a gene coding the factor which inhibits production, function or activity of protein of Cip/Kip family and nucleic acid sequence which inhibits production of protein of Cip/Kip family. There is disclosed pharmaceutical composition containing said vector, and applied for treatment of cardiac diseases. There is offered cardiac hystiocyte produced by the declared method. There is presented method of treating a cardiac disease that implies injection of the disclosed pharmaceutical composition.

EFFECT: regeneration and recovery of cardiac hystiocytes.

31 cl, 13 dwg, 1 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to clinical biochemistry and pertains to a method of extracting extracellular DNA from blood. Nonidet P-40 or triton X-100 is added to the analysed blood sample until final concentration of 0.5 to 1.5%. The mixture is incubated in ice for 1 to 15 minutes. Lysed blood is divided into supernatant and a nuclear fraction of cells by centrifuging. The supernatant is then treated with a RNA-hydrolysing enzyme and extracellular DNA is extracted from the obtained fraction using a fibre-glass sorbent.

EFFECT: use of the method simplifies the procedure of extracting extracellular DNA, simultaneously extract extracellular DNA, which is free and which is bonded to the surface of blood cells, from blood lysate, which allows for significantly increasing reliability and sensitivity of further amplification analysis for early detection of oncological and other diseases.

2 cl, 4 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to substances and methods of treatment, prevention and improvement of the complement-related disease. There are offered various therapeutically effective aptamer/PEG conjugates. Aptamers under the present invention contain modified nucleotides that improves their resistance to enzymatic and chemical destruction, as well as to thermal and physical destruction. These aptamer/PEG conjugates are applicable as agent for therapy of the complement-related cardiac, inflammatory and autoimmune disorders, ischemia reperfusion injury and/or the other diseases or disorders wherein C5-mediated complement activation is involved.

EFFECT: development of the new agent and method of treatment, prevention and improvement of the complement-related disease.

20 cl, 67 dwg, 7 tbl, 6 ex

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