RU2272072C1 - Method for isolation of nucleic acids - Google Patents

Abstract

FIELD: biotechnology, molecular biology, biochemistry.

SUBSTANCE: invention proposes a method for isolation of nucleic acids that involves adsorption of nucleic acids from a sample on fiberglass sorbent treated preliminary with hydrofluoric acid followed by washing out a sorbent from impurities on non-nucleotide nature and elution of nucleic acid being the adsorption process is carried out in the presence of a chaotropic agent. Using the invention provides the enhanced yield of the end product and to decrease duration of process of isolation of nucleic acids. Invention can be used in medicine.

EFFECT: improved isolating method.

10 cl, 2 dwg, 7 ex

Description

The invention relates to the field of biotechnology and molecular biology, and in particular to a method for the isolation and purification of deoxyribonucleic (DNA) and ribonucleic (RNA) acids from biological fluids and other samples.

The production of highly purified nucleic acids (NKs) is an important problem in molecular biology. To obtain NK from biological fluids, methods such as CsCl ₂ gradient centrifugation, gel filtration, phenol treatment, and alcohol precipitation are used. These methods are distinguished by their duration, multi-stage and laboriousness.

The most common way to isolate NK is a method involving the treatment of biological fluid with phenol, followed by precipitation of the selected target product with alcohol [1]. The disadvantages of this method are the duration of the allocation of NK (not less than 3.5 hours due to reprecipitation of the target product), the inconvenience of working with phenol and chloroform, as well as the low yield of low molecular weight product (25-50%).

A known method of RNA isolation, by mixing a sample homogenized in a 4 M solution of guanidine thiocyanate (GTZ) with water-saturated phenol with the formation of a monophasic solution [2]. After the addition of chloroform, phase separation occurs: RNA is in the aqueous phase, while DNA and proteins are associated with the interphase and the organic phase. The disadvantages of this method are the duration of RNA isolation (about 3 hours), the multi-stage process, the inconvenience of working with phenol and chloroform, as well as the low yield of low molecular weight product (7%).

A known method of DNA extraction from clinical samples using finely divided mixed (standardized in size) glass [3]. The method consists in the fact that DNA is adsorbed from a buffer solution containing 4 M GTZ, 0.1 M EDTA (pH 8.0) for 10 minutes, then the silicate sorbent is separated by centrifugation (30 sec at 1000 g), the silicate sorbent is washed twice with adsorption buffer, twice with 70% ethanol and once with acetone. The silicate sorbent is dried for 10 min at 56 ° С, and then the DNA is eluted by incubation with a buffer containing 10 mM Tris-HCl, 10 mM EDTA (pH 8.0) (TE buffer) while heating to 56 ° С for 10 min.

The disadvantages of this method are the duration of DNA extraction (30-40 min), low sorption capacity of the silicate sorbent (0.5 μg of DNA is adsorbed per 1 mg of mixed fine glass). This method allows to isolate DNA with a length of more than 40 TPN, while it is possible to elute from the silicate sorbent no more than 40-50% of the DNA due to the fact that 50-60% of the adsorbed DNA is irreversibly bound to the mixed fine glass. Thus, a DNA yield of at least 40 kbp makes up from 40 to 50%.

A known method of DNA extraction from biological fluids on a silicate sorbent treated with nitric acid [4]. To do this, finely dispersed glass is treated with concentrated HNO ₃ for one hour, then washed with water, the adsorption of DNA to this glass and elution from the silicate sorbent are carried out under the conditions of the analogue described above.

The disadvantages of this method are the duration of DNA extraction (30-40 min), low sorption capacity of the silicate sorbent (0.5 μg of DNA is adsorbed per 1 mg of fine glass). This method allows DNA to be isolated with a length of more than 40 kb, while it is possible to elute from the silicate sorbent no more than 40-50% due to the fact that 50-60% of the adsorbed DNA irreversibly binds to fine glass. Thus, a DNA yield of at least 40 kbp makes up from 40 to 50%.

A known method of separating NK, including adsorption of NK on Aerosil (A-300) in a chaotropic agent, separation of the sorbent and its associated DNA, elution of DNA from Aerosil and purification of the target product. Aerosil is introduced into a solution containing NK to a final concentration of 0.3-0.6%, while a medium with a content of 0.5-2 M GTZ is used to isolate single-stranded DNA and RNA, and a medium with a content of 4 M is used to isolate double-stranded DNA GTZ [5]. The disadvantages of this method are its duration and complexity.

Closest to the claimed prototype method is a method of separating NK, including mixing the initial sample with a buffer solution containing a chaotropic agent, adsorption of NK by filtering the mixture through a glass fiber sorbent (filter GF / F), washing the sorbent and associated NK, elution of NK and purification of the target product [6]. Moreover, to isolate DNA, 350 μl of 4 M GTZ containing 2% sarcosyl is added to 50 μl of a sample resuspended in 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and applied to a GF / F filter, centrifuged for 2-3 min , washed by centrifugation 1 time with 400 μl of 4 M GTZ (without sarcosyl), 2 times with 400 μl of 80% isopropanol, the filters were dried at 37 ° C for 10 min, DNA was eluted with 100 μl of water. To isolate RNA, 300 μl of 4 MHC are added to 300 μl of the sample, incubated at 70 ° C for 2-3 minutes, transferred to a GF / F filter, centrifuged for 5-10 s, washed by centrifugation 2 times with 1 M GTZ (400 μl each) 2 times with 80% isopropanol, the filters are dried at 37 ° C for 10 min, RNA is eluted with 50 μl of water.

The disadvantages of the prototype are the limited functionality of the method, insufficient yield of the target product due to the low sorption capacity of the sorbent. This method allows you to select only high molecular weight NK, while it is possible to elute from the filter no more than 40-50% of high molecular weight NK due to the fact that 50-60% of the adsorbed NK irreversibly binds to the carrier. Thus, the yield of high molecular weight nanocrystals is from 40 to 50%, and nanocrystals with a low molecular weight are released much less efficiently.

An object of the invention is to increase the yield of the target product, reduce the duration of the process, increase the functionality of the method due to the ability to isolate NK molecules of a given length.

The task is achieved by the proposed method, which consists in adsorption of nanocrystals on a glass fiber sorbent pre-treated with hydrofluoric acid in a buffer solution containing a chaotropic agent, washing the sorbent from non-nucleotide biopolymers and subsequent elution of the nanocrystals with a solution containing bicarbonate and EDTA ions, either with distilled water or with buffer solutions with a concentration of not more than 0.1 M. As a result, the resulting supernatant is a solution of purified NK, suitable for molecular biological further studies without additional purification and concentration steps.

The inventive method includes the following sequential stages:

1. Pre-treatment of glass fiber sorbent (SHS) with 0.1-7% hydrofluoric acid for 1-6 hours, followed by washing and drying of the treated SHS (WWS). The obtained OSVS is filled into the tip for a dispenser with a volume of 100-1000 μl (receive a filter); or bake glass wool in a mold at a temperature of more than 500 ° C for 5-30 minutes, then form disks using die-cutting, which are then inserted into test tubes as a filter septum (second filter option). As fiberglass materials, glass wool from M20 brand glass, glass wool from neutral glass, mineral glass wool from basalt glass fiber containing silicon dioxide as the main component are used.

2. Isolation of NK from biological fluids by adsorption on an SIA by filtering a sample through a given sorbent in a buffer solution containing a chaotropic agent, and the isolation of DNA with a length of 500 bp and more carried out in a buffer solution containing 4.5 M GTZ, 20 mm EDTA, 10 mm Tris-acetate, pH 6.4; DNA isolation with a length of 50-500 bp carried out in a buffer solution containing 4.5 M GTZ, 20-70% ethanol, 20 mm EDTA, 10 mm Tris-acetate, pH 6.4, and the selection of RNA is carried out in a buffer solution containing 1 M GTZ, 20-70% isopropanol, 20 mM EDTA, 1.3 mM diethyl pyrocarbonate (DEPC), 10 mM Tris-acetate, pH 4.5.

3. The washing of the SIA and associated NK with a double working buffer solution for sorption of DNA or RNA (containing 4.5 M GTZ, 20 mM EDTA, 10 mM Tris-acetate, pH 6.4 for DNA isolation with a length of 500 bp or more ; 4.5 M GTZ, 20-70% ethanol, 20 mM EDTA, 10 mM Tris-HCl, pH 6.4 to isolate DNA with a length of 50-500 bp or more; or 1 M GTZ, 20 mM EDTA, 1, 3 mM diethyl pyrocarbonate (DEPC), 10 mM Tris-acetate, pH 4.5 to isolate RNA) and twice with a buffer solution containing 60-80% ethanol, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0;

4. Elution of NK with SISF buffer solution containing 5-50 mM bicarbonate ions (HCO ₃ ^- ), 5-10 mM EDTA, pH 8.0-10.0 (preferred option), or with distilled water, or other buffer solutions with a concentration of not more than 0 ,1M.

The following types of filtration can be used for filtering a sample through an SIA and washing it: pressure filtration, vacuum filtration, or centrifugal filtration.

The time of allocation of NK by this method is 10 minutes. The method provides the selection of NK from a length of 100 bp to high molecular fractions from biological samples with high efficiency and does not depend on the concentration of NK in the sample.

In the table. 1 shows the data on the elution of P ³² -radio-labeled DNA (length from 100 to 200 bp) and P ³² -radio-labeled RNA (100 n) with various eluents. From table 1 it is seen that the elution of NK associated with OSVS, a buffer containing bicarbonate and EDTA ions is more effective than a TE buffer and does not depend on the amount of adsorbed DNA. Figure 1 shows the electrophoresis of DNA in a 1% agarose gel containing 0.0003% ethidium bromide. From the first to the fourth track, dilutions of the initial marker of the length of the M13 DNA (from 100 bp to 955 bp) were plotted (Sibenzim, Novosibirsk). On tracks from fifth to eighth, dilutions of the M13 DNA length marker are plotted, after the M13 DNA was isolated by adsorption on the SISF by the claimed method. Figure 1 shows that the proposed method allows you to select both low molecular weight and high molecular weight DNA fragments. Figure 2 shows the electrophoresis of RNA isolated in various ways: 1 - by the claimed method, 2 - by treatment with phenol. RNA was isolated from A431 cells. Figure 2 shows that the inventive method allows more efficient to isolate both low molecular weight and high molecular weight RNA fragments.

Table 2 presents the data on the extraction of P ³² radio-labeled DNA (length from 1000 to 100 bp) and P ^32- radio-labeled RNA (length 100 N.) introduced into human blood plasma by the proposed method. Table 3 presents the data on the isolation of RNA from cells of the A 431 line by treatment with phenol [1], by means of guanidine-phenol extraction [3], as well as on the isolation of RNA by filtration through SIA proposed method. From both tables it can be seen that the inventive method provides almost quantitative selection of NK from biological fluids in significantly less time.

The defining hallmarks of the proposed method, compared with the prototype, are:

1) As a sorbent, glass fiber (containing silicon dioxide as the main component) is used, treated with 0.1-7% hydrofluoric acid solution for 1-6 hours, which allows to increase the working surface and sorption capacity of the latter. OSVS binds both low molecular weight and high molecular weight fragments of NK. From table 2 it can be seen that the capacity of the SIA is 10 times higher than that of untreated SHS. The higher molecular weight DNA fragments of the SISF binds more efficiently (data not shown).

2) For DNA isolation with a length of 500 bp and more use a buffer solution containing 4.5 M GTZ, 20 mm EDTA, 10 mm Tris-acetate, pH 6.4, for DNA extraction with a length of 50-500 bp use a buffer solution containing 4.5 M GTZ, 20-70% ethanol, 20 mM EDTA, 10 mM Tris-acetate, pH 6.4, and to isolate RNA - 1 M GTZ, 20 mM EDTA, 1.3 mM diethyl pyrocarbonate (DEPC ), 10 mM Tris-Acetate, pH 4.5, which allows for quantitative extraction of NK of a given length.

In this case, the sorbent is washed under pressure, using vacuum filtration or by centrifugation, which ensures the separation of nanocrystals in a faster, more technologically advanced and reproducible way. Washing the sorbent with a buffer solution for sorption of DNA or RNA, and then with a buffer solution containing 60-80% ethanol, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0 ensures the effective removal of impurities of a non-nucleotide nature and the absence of NC losses associated with the sorbent. Due to this, the isolated nanocrystals are suitable for molecular biological studies without additional purification steps.

In this case, the elution of nanoparticles associated with the sorbent is predominantly carried out by treating the SISF with a buffer solution containing 5–50 mM bicarbonate ions, 5–10 mM EDTA, pH 8.0–10.0, which allows more rapid and quantitative separation from the sorbent.

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

Example 1

For DNA adsorption, a preliminary treatment of SHS based on neutral glass fiber with hydrofluoric acid with a concentration of 3.5% on a shaker for 4 hours is carried out, the SISF is dried at 200 ° C for 1 hour. Human epidermal carcinoma A431 cells are added to a Petri dish with physiological saline washed 0.5 ml of a mixture containing 4.5 M GTZ, 20 mM EDTA, 10 mM Tris-Acetate pH 6.4, are carefully resuspended and applied to the SIA, filled into the dispenser tip with a volume of 100-1000 μl. Then, with the help of a dispenser, the mixture deposited on the SISF is squeezed (filtered), thereby ensuring the adsorption of DNA on the SISC and the simultaneous removal of the mixture containing no DNA. SIAs are washed from impurities of a non-nucleotide nature twice with a buffer solution containing 4.5 M GTZ, 20 mm EDTA, 10 mm Tris-acetate pH 6.4, and then twice with a buffer solution containing 20% ethanol, 100 mm NaCl, 10 mm Tris-HCl pH 8.0. Elute the DNA with the SISF buffer solution containing 50 mM NaHCO ₃ , 5 mM EDTA at pH 10.0 for 2 min. After centrifugation at 10,000 g for 1 min, the supernatant containing DNA was collected, the pH of the solution was neutralized with 1/10 volume of 100 mM Tris-HCl, pH 7.1.

To assess the completeness of DNA extraction by the claimed method, comparative experiments were performed on the extraction of DNA by precipitation with trichloroacetic acid and adsorption of an acid-insoluble fraction on nylon filters. For this, cells of human epidermal carcinoma A431 were grown on a medium with H ³ -thymidine. H ³ -labeled DNA was isolated from the same number (10 ⁶ ) of cells by the proposed adsorption method on the SIA and the standard method of DNA adsorption on nylon filters. To isolate DNA on the filter, an aliquot of cells (10 ⁶ ) was transferred to a 0.2 μm nylon filter, washed with 0.15 M NaCl and fixed with 5% trichloroacetic acid. The radioactivity of DNA isolated by various methods was measured on a RacBeta 1211 β-scintillation counter. It was found that the radioactivity of H ³ -labeled DNA isolated by various methods was the same. Thus, it was shown that the proposed method provides a quantitative selection of DNA. The DNA content in the sample was determined by measuring the fluorescence of the samples in complex with Hoechst 32258. The fluorescence of DNA complexes with Hoechst 32258 was measured on a Hitachi MPF4 fluorimeter at an excitation wavelength of 350-360 nm and emission of 480-495 nm [5]. To construct the calibration curves, we used a standard solution of fragmented DNA from calf thymus at concentrations of 12.5, 25, 50, 100, 200, 400 ng / ml. In this way, 10 μg of DNA was isolated from 2 × 10 ⁶ cells of line A 431. The time of DNA extraction from samples by this method is 8-10 minutes. The DNA binding capacity of the glass is up to 2.3 μg DNA per 1 mg SBS.

Example 2

For DNA adsorption, M20 glass wool was pretreated by incubation with 7% hydrofluoric acid on a rocking chair for 2 hours, washed, then baked in a mold at 600 ° C for 15 minutes, a disk of the required diameter was formed by die cutting and placed it in a test tube (received a filter with a partition from the SIS).

0.5 ml of a mixture containing 4.5 M GTZ, 20 mM EDTA, 10 mM Tris-acetate, pH 6.4 was added to a Petri dish with washed saline cells, carefully resuspended and then applied to the SIS. Then the samples were centrifuged for 1 min at 300 g. The SIA was washed twice with a buffer solution containing 4.5 M GTZ, 20 mM EDTA, 10 mM Tris-acetate, pH 6.4, and then twice with a buffer solution containing 60% ethanol, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0. DNA was eluted with an SIA of 5 mM NaHCO ₃ , 10 mM EDTA at pH 8.0 for 2 min. After centrifugation at 10,000 g for 1 min, the supernatant containing DNA was collected, the pH of the solution was neutralized by 1/10 volume of 100 mM Tris-HCl, pH 7.1. The time of DNA extraction from samples by this method is 8-10 minutes.

Example 3

To 0.5 ml of human blood plasma was added 1 ml of a mixture containing 4.5 M GTZ, 20 mM EDTA, 10 mM Tris-acetate, pH 6.4, carefully resuspended and then applied to the SIA obtained analogously to example 1. Procedure for isolation and determination DNA concentrations using fluorescence of DNA complexes with Hoechst 32258 were carried out analogously to examples 1 and 2. The concentration of DNA in the blood plasma of healthy donors was 20 ng / ml plasma.

Example 4

For RNA adsorption, basalt fiberglass was pretreated with 0.4% hydrofluoric acid on a shaker for 3 hours, then the SISF was baked in a mold at 650 ° C for 10 min, disks of the required diameter were cut out and placed in test tubes. Cells of human epidermal carcinoma A431 were used to isolate RNA. Cells were cultured in DMEM containing 10% fetal calf serum in an atmosphere of 5% CO ₂ at 37 ° C. A 0.25% trypsin solution was used to dissociate the grown cells. After neutralizing trypsin, the cells were transferred to tubes and pelleted by centrifugation at 2500 g for 5 min. The precipitated cells were resuspended in 20 μl of physiological saline and 60 μl of lysis buffer containing 0.6% Nonidet P40, 50 mM Tris-HCl, 0.15 M NaCl, 5 mM NaF, 1 mM PMSF, 2 mM EDTA, 1 mM Na ₃ MoO _{4 was} added. . Cells were incubated for 15 min and centrifuged for 10 min at 14,000 rpm (Eppendorf5410 centrifuge) to separate the membrane-cytosolic and nuclear fractions. The supernatant containing the membrane-cytosolic fraction was transferred to other tubes and 1 ml of buffer containing 1 M GTZ, 2 mM DEPC, 20% isopropanol, 20 mM EDTA, 10 mM Tris-acetate pH 4.5 was added. The mixture was vortexed vigorously for 10 seconds and then applied to the SIS. Pressure filtration was used to filter the sample through the sorbent and wash the sorbent. The supernatant was removed, the sorbent was washed twice with a buffer solution containing 1 M GTZ, 20 mM EDTA, 2 mM DEPC, 10 mM Tris-acetate pH 4.5, 20% isopropanol, and then twice with a buffer solution containing 80% ethanol, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0. RNA with SISF was eluted with a buffer solution containing 40 mM NaHCO ₃ , 7 mM EDTA, pH 9.0, for 2 min. After centrifugation at 10,000 g for 1 min, the supernatant containing RNA was collected, the solution was neutralized by 1/10 volume of 100 mM Tris-HCl to pH 7.1.

The RNA content in the sample was determined spectrophotometrically. As a result, 28 μg of RNA was isolated from 1 × 10 ⁶ cells of the A 431 line. The sample preparation time with subsequent RNA isolation was 20 min.

Example 5

To adsorb RNA, neutral glass fiber was pretreated with 0.1% hydrofluoric acid by incubation on a rocking chair for 6 hours. The sorbent was made by baking in a mold at 550 ° C for 20 min. The processing of A-431 cells to obtain a membrane-cytosolic fraction was carried out analogously to example 4. Next, the supernatant containing the membrane-cytosolic fraction was transferred to other tubes, 1 ml of buffer containing 1 M GTZ, 2 mM DEPC, 20 mM EDTA, 70 was added % isopropanol, 10 mM Tris-acetate pH 4.5. The mixture was vortexed vigorously for 10 seconds and then applied to the SIS. Vacuum filtration was used to filter the sample through the sorbent and wash the sorbent. The SIA was washed twice with a buffer solution containing 1 M GTZ, 2 mM DEPC, 20 mM EDTA, 40% isopropanol, 10 mM Tris-Acetate pH 4.5, and then twice with a buffer solution containing 75% ethanol, 100 mM NaCl, 10 mM Tris- HCl, pH 8.0. RNA was eluted with SISR with distilled water for 5 minutes. After centrifugation at 10,000 g for 1 min, the supernatant containing RNA was collected.

The RNA content in the sample was determined spectrophotometrically. As a result, 20 μg of RNA was isolated from 1 × 10 ⁶ cells of the A 431 line. The time for RNA isolation from the supernatant containing the membrane-cytosolic fraction was 10 min.

Example 6

To 0.2 ml of human blood plasma was added 2 ml of a buffer solution containing 1 M GTZ, 2 mM DEPC, 20 mM EDTA, 70% isopropanol, 10 mM Tris-acetate pH 4.5. The mixture was vortexed vigorously for 10 seconds and applied to the SIA. The RNA isolation procedure was carried out similarly to examples 4 and 5. The RNA content in the sample was determined by measuring the fluorescence of the samples in combination with SYBR Green II. The fluorescence of RNA complexes with SYBR Green was measured on a Bio-Rad fluorimeter at an excitation wavelength of 490 nm and emission of 520 nm [5].

Example 7

For DNA adsorption of 100 bp in length preliminarily processed neutral carbon fiber based CB С by incubation with 0.1% hydrofluoric acid on a rocking chair for 2 hours. The SIA was dried at 200 ° C for 1 hour. The PCR reaction products were dispersed in a 1% agarose gel containing 0 , 0003% ethidium bromide, then DNA was visualized under a UV emitter and the desired DNA fragment of 100 bp was cut out. A piece of agarose was placed in a test tube and incubated for 10 min at 65 ° C. To molten agarose (0.1 ml) was added 1 ml of a mixture containing 4.5 M GTZ, 20 mM EDTA, 70% ethanol, 10 mM Tris-acetate, pH 6.4, carefully resuspended and applied to the SIA filled in a 100- dispenser tip 1000 μl Then, the mixture deposited on the SISF was pressed through with a dispenser. The SIA was washed twice with a buffer solution containing 4.5 M GTZ, 20 mm EDTA, 50% ethanol, 10 mm Tris-acetate, pH 6.4, and then twice with a buffer solution containing 80% ethanol, 100 mm NaCl, 10 mm Tris-HCl, pH 8.0. DNA with SIA was eluted with a TE buffer for 2 minutes. After centrifugation at 10,000 g for 1 min, the supernatant containing DNA was collected.

Using the proposed method will allow, in comparison with the prototype:

- reduce media consumption; reduce the cost of the allocation of NK;

- increase the efficiency of NC extraction by 10 times due to the effective sorption and elution of the adsorbed NC on the carrier;

- to increase the functionality of the method by providing the ability to highlight both low molecular weight and high molecular weight fragments of NK, as well as NK of a given length;

- reduce the time of the isolation procedure to 8-10 minutes;

- increase the sensitivity of PCR - and RT - PNR - diagnostic systems due to the binding of low molecular weight fragments of NK;

- to provide the possibility of using the proposed method for the quantitative determination of the concentration of NK in biological fluids;

- reduce the amount of biological material used to isolate NK for the purpose of subsequent amplification or other research.

INFORMATION SOURCES

1. Short protocols in molecular biology. Third edition. Wiley, 1995.

2. Chomezynski P., Mackey K. Single-step method of total RNA isolation by acid Guanidine-Phenol extraction // Organelles and cellular structures. 1996. V.10. P.221-224.

3. Beld M., Sol C., Goudsmit Fractionation of nucleic acids into single-stranded and double-stranded forms // Nucl. Acids Res. 1996. V.24. P.2618-2619.

4. Short protocols in molecular biology. Third edition. Wiley, 1995.

5. RF patent No. 2119954, cl. C 12 N 15/10, C 07 H 21/04, publ. 10/10/98.

6. Gribanov O., Scherbakov A., Perevozchikova I., Gusev A. Use of aerosil and GF / F filters to clean DNA fragments, plasmid DNA and RNA // Biochemistry 1996. V.61. S.10.

Table 1 The number of NK introduced into the sample, ng / ml The percentage of extracted NK, ng / ml WWS SHS Elution 50 mM NaHCO ₃ , 5 mM EDTA, pH 8.0 Elution 0.01 M TE buffer Elution 0.01 M TE buffer DNA 10 82 48 - 25 85 48 fifteen fifty 84 fifty 17 one hundred 86 51 16 200 87 53 17 RNA 10 75 40 - 25 77 41 - fifty 79 44 fifteen one hundred 80 49 fourteen 200 81 fifty fifteen table 2 Fiberglass sorbent Not processed Treated 0.1-7% HF 1 hour 2 h 3 h 4 h 5 h 6 h DNA 8 ± 3 82 ± 5 84 ± 5 83 ± 4 85 ± 4 87 ± 3 86 ± 3 RNA 7 ± 5 76 ± 5 78 ± 5 79 ± 6 77 ± 5 80 ± 6 78 ± 5 Table 3 Allocation Method Allocation time (hours) RNA isolation efficiency Low molecular weight ³² P-labeled RNA (100n.),% High molecular weight RNA isolated from 10 ⁶ cells,% (μg) Phenolic four 25 81 (26) Phenol-guanidine 2,5 7 39 (13) On the SIA 0.2 55 100 (28)

Claims (8)

1. A method for isolating nucleic acids, comprising mixing a sample with a buffer solution containing a chaotropic agent, adsorption of nucleic acids on a glass fiber sorbent, washing it from impurities of a non-nucleotide nature, and eluting the nucleic acids, characterized in that the sorbent is pre-treated with 0.1-7.0 % hydrofluoric acid solution for 1-6 hours 2. The method according to claim 1, characterized in that for the isolation of DNA with a length of 50-500 bp the adsorption of the latter is carried out in a buffer solution containing 4.5 M GTZ, 20-70% ethanol, 20 mm EDTA, 10 mm Tris-acetate, pH 6.4. 3. The method according to claim 1, characterized in that for the isolation of DNA with a length of 500 bp and more, the adsorption of the latter is carried out in a buffer solution containing 4.5 M GTZ, 20 mm EDTA, 10 mm Tris-acetate, pH 6.4. 4. The method according to claim 1, characterized in that for the isolation of RNA, the adsorption of the latter is carried out in a buffer solution containing 1 M GTZ, 20-70% isopropanol, 20 mm EDTA, 1.3 mm DEPC, 10 mm Tris-acetate, pH 4 ,5. 5. The method according to claim 1, characterized in that glass fiber sorbent is used glass wool from glass brand M20, glass wool from neutral glass, mineral glass wool from basalt glass fiber containing silicon dioxide as the main component. 6. The method according to claim 1, characterized in that the washing of the sorbent is carried out under pressure, under vacuum or by centrifugation. 7. The method according to claim 1, characterized in that the washing of the sorbent from impurities of non-nucleotide nature is carried out first twice with a working buffer solution for sorption of DNA or RNA, and then twice with a buffer solution containing 60-80% ethanol, 100 mm NaCl, 10 mm Tris -HCl, pH 8.0 8. The method according to claim 1, characterized in that the elution of nucleic acids is preferably carried out with a buffer solution containing 5-50 mM bicarbonate ions, 5-10 mM EDTA at pH 8.0-10.0, or with distilled water, or diluted buffer solutions with a molarity of not more than 0.1.

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