RU2696052C1 - Method for extracting dna from soil - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology and molecular biology and is intended for analysis of genetic material of soil microorganisms in order to study their diversity and when designing genetically engineered producer strains. To achieve the technical result, the soil sample is homogenized by vibration in the presence of grinding bodies in a solution containing guanidine HCl, sodium phosphate buffer, TrisHCl, sodium dodecyl sulphate in amount of 1–4 wt%, sodium lauryl sarcosinate in amount of 2–6 wt%, phenol-chloroform mixture is added, followed by centrifugation and isopropanol DNA deposition. Method provides yield of DNA of order of 10,000 ng per 0.2 g of chernozem.EFFECT: high output of metagenomic DNA from soil owing to higher efficiency of cell destruction and desorption of biopolymers.3 cl, 2 tbl

Description

The invention relates to biotechnology and molecular biology, and is intended for the analysis of the genetic material of soil microorganisms in order to study their diversity and in the design of genetically engineered producer strains. The method provides a DNA yield of the order of 10,000 ng per 0.2 g of chernozem.

Over the past two decades, revolutionary changes have occurred in all branches of biological science related to microbiology - scientists have come to the conclusion that the biodiversity of microorganisms is strikingly superior to the ideas that have been formed over the past 100-150 years. It turned out that less than 1% of all existing microorganisms were previously studied, and approximately 99% cannot currently be cultivated in laboratory conditions and, accordingly, have not been subjected to any study. At the moment, methods are being developed that are not related to the production of crops, and allowing the study of microorganisms within their natural habitat. Such methods are called metagenomic. Metagenomics is a branch of molecular genetics that studies “metagenomes” - genetic material obtained directly from samples taken from the environment. Traditional genome sequencing relies on cultured culture clones, while metagenomics works with a set of all the DNA in the sample. The main difference when using the metagenomic approach is the count of uncultured microorganisms, along with cultivated ones.

The choice of DNA extraction method is one of the most important stages in the study, since the success of further studies depends on the quality and quantity of DNA extracted. Existing methods for isolating soil metagenomic (total) DNA often do not allow obtaining enough DNA for research (metagenomic sequencing) of the required purity. It should be noted that at the initial stages of DNA extraction from the soil, it is necessary to obtain the maximum possible number of nucleic acids, since in the process of further purification the DNA loss can be significant. Known methods for the extraction of DNA from the soil, mainly differ in the use of various surface-active substances (surfactants) in order to achieve the best destruction of cells, dissociation of biopolymers and their desorption from soil particles. The DNA yield when using these methods, as a rule, is small, which leads to an increase in the processed soil volume, an increase in labor costs and reagent consumption. In this regard, researchers are constantly making attempts to increase the yield of DNA; various methods and reagent kits are proposed.

Commercial reagent kits for isolating metagenomic soil DNA are known. DNA isolation in this case is carried out in accordance with the manufacturers protocol. Kit for isolating genomic DNA from soils of FastDNA Spin Kit For Soil (MP Biomedicals) (FastDNA Spin Kit For Soil, MP Biomedicals. Electronic resource: https://www.dia-m.ru/lab/gomogenizatory-fastprep/acs/17086 /) [1] used with the FastPrep homogenizer or with an alternative appliance. Up to 500 mg of the soil sample is placed in 2 ml tubes containing the lysing matrix E - a mixture of glass (4 mm in diameter), quartz (0.1 mm in diameter) and ceramic (1.4 mm in diameter) balls capable of destroying all soil organisms. Homogenization occurs in the presence of MT buffer and sodium phosphate. Upon completion of the lysis, the samples are centrifuged. The resulting DNA is purified using Geneclean technology, including the use of SPIN silica gel filters and elimination of humic acids and polyphenols.

However, this kit has a high cost, which is about $ 400 per 50 secretions, the DNA yield is low and is about 1-5 μg from 500 mg of soil [1].

Also known is the NucleoSpin® Soil kit (MACHEREY-NAGEL) (Genomic DNA from soil. User manual. NucleoSpin® Soil. November 2017 / Rev. 07. http://www.mnnet.com/Portals/8/attachments/Redakteure_Bio/Protocols /Genomic%20DNA/UM_gDNASoil.pdf) [2], which is designed to isolate total DNA from the soil. The kit includes two alternative lyse buffers and an Enhancer SX supplement that can be combined with both lyse buffers. For quantitative DNA binding, optimized silica columns — NucleoSpin® Soil Columns — are used. NucleoSpin® Bead Tubes containing ceramic beads that allow the most efficient lysis of microorganisms in the sample. For convenient removal of contaminants, NucleoSpin® Inhibitor Removal Columns are included in the kit. Initially, the sample material is resuspended in SL1 or SL2 lysis buffer supplemented with Enhancer SX and mechanically destroyed using ceramic beads. Proteins and polymerase chain reaction inhibitors (PCR) are precipitated with SL3 lysis buffer and then precipitated by centrifugation together with ceramic beads and a sample of material. The supernatant (supernatant) was collected and purified by passing it through a NucleoSpin® Inhibitor Removal Column column to remove inhibitors. DNA binding is regulated by the addition of SB buffer and the lysate is loaded onto a NucleoSpin® Soil Column column. Residual humic substances, especially humic acids and other PCR inhibitors, are removed by efficient washing with SB buffer and washing buffers SW1 and then SW2. After the drying step, ready-to-use DNA can be eluted (washed away) with SE SEPLUS buffer - 5 mM Tris / HCl, pH 8.5.

This kit has a high cost, which is approximately $ 370 per 50 precipitates and a low DNA yield of about 2-10 μg from 0.5 g of soil [2, C. 7].

A known kit for DNA isolation (RU 2650865, IPC C12N 15/10, publ. 04/17/2018) [3], including lyse, wash and elution buffer solutions. The lysis buffer solution contains guanidine thiocyanate, Tris hydrochloride, triton and sorbent in the form of a suspension of magnetic microspheres in saline. Wash buffer No. 1 contains guanidine thiocyanate, Tris hydrochloride and ethyl alcohol. Wash Buffer No. 2 contains Tris hydrochloride, sodium chloride, and ethyl alcohol. The eluting solution is deionized water. A known set of reagents for DNA isolation has a small yield of DNA - from 200 to 500-1000 ng from 5 g of the starting material [3, C. 3].

In order to facilitate the exit of DNA from cells, in addition to certain reagents, methods using physical factors of influence are used.

For example, in a method for isolating DNA from soil (Bintrim SB, Donohue TJ, Handelsman J., Roberts GP, Goodman RM Molecular phylogeny of Archaea from soil // Proc. Natl. Acad. Sci. USA. - 1997.- V. 94 ( 1). - P. 277-282. Https://doi.org/10.1073/pnas.94.L277) [4, C. 277-278] used ultrasound to facilitate the release of DNA from cells. 500 milligrams of soil are resuspended in 500 μl of solution A containing 250 mM NaCl and 100 mM Na ₂ EDTA (disodium salt of ethylenediaminetetraacetic acid) and sonicated in a Branson 2200 ultrasonic bath for 3 minutes. Then lysozyme is added to a concentration of 0.5 mg / ml and the mixture is incubated at 37 ° C for 30 min with periodic stirring. Proteinase K is then added to a final concentration of 2.0 mg / ml and the mixture is incubated for another 30 minutes. After incubation, 500 μl of solution B, which contains 250 mM NaCl, 100 mM Na ₂ EDTA, 4% (w / v) sodium dodecyl sulphate (sodium dodecyl sulphate, SDS), and 75 μl of 5 M guanidine isothiocyanate are added and the mixture is gently mixed. The mixture was incubated at 68 ° C for 1 hour with periodic stirring. After incubation, the sample is mixed in a 1: 1 ratio with zirconia / silica pellets with a diameter of 0.1 mm and homogenized in a Mini-Beadbeater (type BX-4, Biospec Products, USA) at 3000 rpm for 45 seconds. Samples were centrifuged to remove granules and the supernatant was taken, to which 150 microliters of cetyltrimethylammonium bromide (CETyltrimetylammonium bromide, CTAB) solution was added, which contained 2% (w / v) CTAB, 100 mM Tris⋅HCl pH 8.0, 20 mM Na ₂ EDTA, 1.4 M NaCl, and stirred. The mixture was incubated at 65 ° C for 15 min with periodic stirring and then sequentially extracted with equal volumes of a mixture of chloroform: isoamyl alcohol (24: 1), phenol: chloroform: isoamyl alcohol (24: 24: 1) and chloroform: isoamyl alcohol (24 :one). An equal volume of isopropanol is added to the supernatant, and total DNA is recovered by centrifugation. DNA is resuspended in 500 μl of 10 mM Tris⋅HCl (pH 8.0) and, for amplification, PCR is purified by 4-fold ultrafiltration using Microcon-100 microconcentrators (Amicon).

The disadvantage of this method is the complexity, and the use of ultrasound can cause damage to the DNA structure (Grokhovsky SL Specificity of DNA cleavage by ultrasound // Molecular Biology. - 2006. - V. 40 (2). - P. 276-283; Nechipurenko Yu.D. , Golovkin MB, Nechipurenko D.Yu., Ilyicheva I.A., Panchenko L.A., Polozov R.V., Grokhovsky S.L. Characteristic features of DNA cleavage by ultrasound // Journal of Structural Chemistry. - 2009. - T. 50, No. 5. - S. 1045-1052) [5, P. 277; 6, S. 1050-1051].

A known method of DNA extraction, in which a soil sample after mixing with a lysis solution containing NaCl, SDS, is incubated at a temperature of 72 ° C for 45 min for lysis of bacterial cells (Sagar K., Singh S., Goutam KK, Konwar B.K Assessment of five soil DNA extraction methods and a rapid laboratory-developed method for quality soil DNA extraction for 16S rDNA-based amplification and library construction. // J Microbiol. Methods. - 2014. - V. 97. - P. 68- 73. doi: 10.1016 / j.mimet.2013.11.008) [7]. The sample is then centrifuged at an acceleration of 13000 g for 5 min at 4 ° C and the supernatant is transferred into a 2-ml centrifuge tube. 100 μl of 6 M potassium acetate and 400 μl of 50% polyethylene glycol (PEG) are added to the supernatant and the mixture is left to precipitate for 20 minutes at -20 ° C and then centrifuged at 4 ° C for 5 minutes. The supernatant is removed and the precipitate is dried in air. Then it is dissolved in 500 μl of TE-buffer (Tris-EDTA buffer) pH 8.0, 500 μl of chloroform is added and centrifuged with an acceleration of 13000 g at 4 ° C for 5 min. Chloroform extraction was repeated twice and 500 μl of isopropanol was added to the supernatant. It is then left to precipitate an aqueous fraction of DNA for 5 min at 4 ° C and again centrifuged at an acceleration of 13000 g for 5 min. The DNA pellet was suspended in 100 μl of 1 × TE (10 mMTris-HCl, 1 mM EDTA (ethylenediaminetetraacetic acid)).

The disadvantage of this method is the low DNA yield, which is 3.8 μg / g of soil [7, C. 70], which may be due to an ineffective cell lysis procedure.

Another method for extracting DNA from soil for cell lysis uses glass powder (Devi SG, Fathima AA, Radha S., Arunraj R., Curtis WR, Ramya M. Rapid and economical method for efficient DNA extraction from diversesoils suitable for metagenomic applications / / PLoS One. - 2015. - V. 10 (7): e0132441. Doi: 10.1371 / journal.pone.0132441) [8]. To isolate DNA, 1 g of a soil sample and 1 g of sterile powder from ground laboratory glass are placed in a sterile mortar and ground for about 5 minutes. For DNA extraction, add 1 ml of 100 mM Tris buffer, 100 mM EDTA, 1.5 M NaCl (pH 8.0) and 10 mg of powdered activated carbon and mix several times by pipetting. Transfer the mixture to a 2 ml tube. Incubate the tube at 65 ° C for 10 minutes in a water bath, and then centrifuged at 12,000 g for 5 minutes at 4 ° C. Transfer 500 μl of the supernatant to a new 2 ml microcentrifuge tube. To the resulting supernatant was added 100 μl of sodium acetate, pH 5.2, and 400 μl of 30% PEG (MW-8000). Allow the mixture to cool at -20 ° C for 20 minutes in the freezer. Test tubes are thawed slowly and then centrifuged at 12,000 g for 5 minutes at 4 ° C. Remove the supernatant and resuspend the pellet with 500 μl of TE-buffer 10 mM Tris, 1 mM EDTA pH 8.0. An equal volume (500 μl) of a mixture of chloroform: isoamyl alcohol, prepared in a ratio of 24: 1, was added. Centrifuged at 12000 g for 5 minutes at 4 ° C. Transfer the aqueous phase to a new tube and add 500 μl of ice-cold isopropanol. Precipitation is carried out for 5 min at 4 ° C and centrifuged at an acceleration of 12,000 g for 10 min at 4 ° C. Remove the supernatant and wash the precipitate with 70% ethanol. Centrifuged at 12000 g for 2 minutes at 4 ° C. The supernatant is removed, the precipitate is dried in air and dissolved in 100 μl of TE buffer (pH 8.0).

The disadvantage of the method is also a low DNA yield equal to 5.48 μg / g of soil [8, C. 9].

A known method of DNA extraction from biological objects on subject media - gauze, paper, synthetic tissues, in which N-lauryl sarcosinate Na is used as a surface-active substance to destroy cells and dissociate biopolymers, and ethylenediaminetetraacetic acid disodium salt to prevent degradation of nucleic acids ( EDTA) (RU 2485178, IPC C12N 15/10, В82В 1/00. Publ. 06/20/2013) [9]. According to the known method, a biological object is ground and placed in a test tube with a lysis buffer solution of the composition: 0.1 M Tris-HCl, 0.1 M EDTA, 0.1 M NaCl, 0.5% N-laurylsarcosyl Na and proteinase K, pH 6-7. A cell lysate is obtained, to which a sorbent of magnetic nanoparticles modified with chitosan is added, mixed and incubated for 25-35 minutes. The test tube is placed on a magnetic tripod, the mixture is divided into fractions - the sorbent associated with DNA and the supernatant. The supernatant is removed, and an elution buffer solution of 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA is added to the precipitate, incubated, the tubes are placed on a magnetic stand, the mixture is separated into sorbent - precipitate and supernatant - DNA fractions dissolved in eluting buffer solution. The precipitate is removed, DNA remains in the supernatant. The product yield is up to 200 ng of DNA from 5 g of bone powder. The method allows to isolate DNA from biological materials, for example, muscle tissue, horns, animal hair, nail plates.

The closest in technical essence to the claimed invention is a method of DNA extraction from soil samples (DNA extraction from soil samples: Guidelines. Russian Academy of Agricultural Sciences. All-Russian Scientific Research Institute of Agricultural Microbiology. - St. Petersburg, 2011. - 27 pp.) [10 , S. 15-17], in which sodium dodecyl sulfate is used as the main detergent for DNA isolation. To a sample of 0.2 g of frozen soil placed in a test tube, add a number of glass beads, approximately equal in volume to the soil, and add a solution of guanidine - guanidine isothiocyanate 240 mM, sodium phosphate buffer 200 mM, pH 7.0, and 350 μl of a 1% SDS - solution TrisHCl 500 mM, SDS 1% (w / v), pH 7.9, and 400 μl phenol-chloroform mixture. The tube is placed in a shaker and the sample is homogenized for 1-15 minutes, depending on the power of the device (FastPrep 24-1 min at maximum power, Vortex Genie® 2-15 min at maximum speed). Then centrifuged at an acceleration of 10-15 × 10 ³ g at maximum speed for 5 minutes The aqueous phase was taken, 400 μl of chloroform was added, centrifuged as in the previous step, and the aqueous phase was taken. An equal volume of isopropyl alcohol was added to the crude DNA extract, centrifuged at maximum speed for 5 minutes, washed with 70% (v / v) ethanol, slightly dried in air and the residue was dissolved at 65 ° C for 5-10 minutes in 100 μl of water.

The methodological instructions do not present the DNA yield. In this regard, the authors of the claimed invention implemented a prototype method and obtained a DNA yield of 1750 ng from 0.2 g of a chernozem sample (Table 2), which is not enough for metagenomic studies, for example, for NGS sequencing (next generation sequencing).

The task of the invention is to increase the yield of metagenomic DNA from the soil by increasing the efficiency of the processes of cell destruction and desorption of biopolymers.

The specified technical result is achieved in that the method of DNA extraction from the soil involves the homogenization of a soil sample by vibration in the presence of grinding media in a solution containing guanidine HCl, sodium phosphate buffer, TrisHCl, sodium dodecyl sulfate (SDS), a phenol-chloroform mixture followed by centrifugation and DNA precipitation with isopropanol.

According to the invention, the solution for soil homogenization additionally contains detergent sodium lauryl sarcosinate in the following ratio of starting components, weight. %:

sodium lauryl sarcosinate - 2-6

sodium dodecyl sulfate - 1-4

In a preferred embodiment:

- homogenization of the soil sample is carried out using a vibrating mill in the frequency range 25-35 Hz;

- as grinding media, a mixture of glass beads with a diameter of 0.5 mm and 1.0 mm and ceramic beads of 2.0 mm in a weight ratio of 3: 1: 1, respectively, is used.

Sodium lauryl sarcosinate C ₁₅ H ₂₈ NNaO ₃ and sodium dodecyl sulfate are surfactants that are used as detergents in industry, pharmacology, and cosmetology (Sodium lauryl sarcosinate. Electronic resource: https://dic.academic.ru/dic.nsf/ruwiki / 1541358 (accessed date: 08/03/2018); Sodium lauryl sulfate. Electronic resource: http://moepravo.guru/vozvrat-i-obmen/obsshaya-informatsiya/sostav/laurilsulfat-natriya (accessed date: 08/03/2018) [ 11, 12].

The combination of known detergents in the claimed concentrations leads to an increase in the efficiency of the processes of cell destruction and desorption of biopolymers due to the synergistic effect - and, as a result, a high yield of DNA from the soil of the order of 10,000 ng, which was not achieved in the prototype and known analogues.

The method is as follows.

In a 2 ml test tube with a screw cap, weighed 0.2 g of frozen soil, to which are added glass diameter 0.5 mm and 1.0 mm and ceramic diameter 2.0 mm beads. Then, 350 μl of a solution of guanidine — guanidine HCl 240 mM, sodium phosphate buffer 200 mM, pH 7.0; 350 μl of a solution of SDS - TrisHCl 500 mM, SDS 1-4%, weight. %, pH 7.9; 350 μl of sodium lauryl sarcosinate solution 2-6%, weight. %, as well as 400 μl of a mixture of phenol-chloroform. The mixture is shaken in a Mixer Mills MM400 mill (Retsch, Germany) for 15 minutes at a frequency of 30 Hz, then centrifuged in a MiniSpin Plus Eppendorf centrifuge for 7 minutes at an acceleration of 14,000 g. The aqueous phase is taken and 400 μl of chloroform is added to it. It is then vigorously shaken and centrifuged for 7 minutes at an acceleration of 14,000 g, then 500 μl of the aqueous phase is taken and 500 μl of isopropyl alcohol is added to it. It is kept in the refrigerator for 15 minutes. Then centrifuged for 7 minutes with an acceleration of 14000 g. The precipitate was washed 2 times with 70% ethanol (v / v) and dissolved in 1X TE buffer. The purity of all reagents used in the experiments is “analytical grade”. DNA concentration was determined by fluorimetric method using a Quibit 3.0 fluorimeter (Termo Fisher Scientific).

Table 1 shows the amount of DNA in ng isolated from a 0.2 g soil sample using sodium dodecyl sulfate and sodium lauryl sarcosinate in the claimed concentrations and beyond.

Table 2 shows the amount of DNA, ng, isolated from a soil sample weighing 0.2 g, obtained according to the claimed method and the prototype method.

Example 1. Isolation of DNA using 0.5% SDS together with a solution of guanidine chloride. Spent DNA extraction according to the prototype method [10], characterized in that the concentration of SDS was reduced to 0.5%.

Example 2. Isolation of DNA according to the prototype method [10].

A 2 ml sample with a screw cap was weighed 0.2 g of frozen soil. An amount of a matrix of glass beads was added, approximately equal in volume to the soil, after which 350 μl of a solution of guanidine - guanidine isothiocyanate 240 mM, sodium phosphate buffer 200 mM, pH 7.0, and 350 μl 1% SDS - TrisHCl 500 mM were added to each eppendorf , SDS 1% (w / v), pH 7.9, as well as 400 μl of a phenol-chloroform mixture. The tube was placed in a shaker and the sample was destroyed for 1-15 minutes, depending on the power of the device (FastPrep 24 - 1 min at maximum power). Then it was centrifuged at an acceleration of 10-15 × 10 ³ g (maximum speed, standard rotor for 1.5 ml Eppendorf tubes) for 5 min. The aqueous phase was carefully selected, 400 μl of chloroform was added, vigorously shaken for 1-3 min, centrifuged at an acceleration of 10-15 × 10 ³ g, the aqueous phase was carefully selected. One volume of isopropyl alcohol was added to the crude DNA extract, shaken vigorously, centrifuged (maximum speed, 5 min), washed with 70% (v / v) ethanol, slightly dried in air and the residue was dissolved at 65 ° C for 5-10 min in 100 μl of water. DNA concentration was determined by fluorimetric method using a Quibit 3.0 fluorimeter (Termo Fisher Scientific).

Example 3. Isolation of DNA using 2% SDS. DNA isolation was performed according to Example 2, characterized in that the concentration of SDS was increased to 2%.

Example 4. Isolation of DNA using 4% SDS together with a solution of guanidine chloride. DNA isolation was carried out according to example 2, characterized in that the concentration of SDS was increased to 4%.

Example 5. Isolation of DNA using 6% SDS together with a solution of guanidine chloride. DNA isolation was carried out according to example 2, characterized in that the concentration of SDS was increased to 6%.

Example 6. Isolation of DNA using 1% sodium lauryl sarcosinate instead of guanidine chloride and SDS.

DNA isolation was performed according to the following procedure.

A sample of 0.2 g of frozen soil was taken into a 2 ml test tube with a screw cap. A quantity of matrix - glass beads was added, approximately equal in volume to the soil, after which 350 μl of 200 mM sodium phosphate buffer and 350 μl of 1% sodium lauryl sarcosinate solution - TrisHCl 500 mM, sodium lauryl sarcosinate 1% (weight / vol) were added to each eppendorf , pH 7.9, as well as 400 μl of a mixture of phenol-chloroform. The mixture was shaken in a Mixer Mills MM400 mill (Retsch, Germany) for 15 minutes at a frequency of 30 Hz, then centrifuged for 7 minutes at an acceleration of 14,000 g. The aqueous phase was taken, 400 μl of chloroform was added to it, and shaken vigorously for 2 minutes. It was centrifuged as in the previous step, then 500 μl of the aqueous phase was taken and 500 μl of isopropyl alcohol was added to it. It was kept in the refrigerator for ~ 15 minutes, after which it was centrifuged for 7 minutes at an acceleration of 14000 g. The precipitate was washed twice with 70% ethanol and dissolved in deionized water. DNA concentration was determined by fluorimetric method using a Quibit 3.0 fluorimeter (Termo Fisher Scientific).

Example 7. Isolation of DNA with 2% sodium lauryl sarcosinate.

DNA isolation was carried out according to example 6, characterized in that the concentration of sodium lauryl sarcosinate was increased to 2%.

Example 8. DNA Isolation Using 4% Sodium Lauryl Sarcosinate

DNA isolation was carried out according to example 6, characterized in that the concentration of sodium lauryl sarcosinate was increased to 4%.

Example 9. DNA Isolation Using 6% Sodium Lauryl Sarcosinate

DNA isolation was carried out according to example 6, characterized in that the concentration of sodium lauryl sarcosinate was increased to 6%.

Example 10. DNA Isolation Using 8% Sodium Lauryl Sarcosinate

DNA isolation was carried out according to example 6, characterized in that the concentration of sodium lauryl sarcosinate was increased to 8%.

Example 11. Isolation of DNA using 4% sodium lauryl sarcosinate together with 0.5% SDS and a solution of guanidine chloride.

A sample of 0.2 g of frozen soil was taken into a 2 ml test tube with a screw cap. An amount of a matrix of glass beads was added, approximately equal in volume to the soil, after which 350 μl of a guanidine solution — guanidine HCl 240 mM, sodium phosphate buffer 200 mM, pH 7.0, and 350 μl of a solution of 0.5% SDS with 4% lauryl sarcosinate were added to each eppendorf sodium - TrisHCl 500 mM, SDS 1% (w / v), sodium lauryl sarcosinate 4% (w / v), pH 7.9, as well as 400 μl of a phenol-chloroform mixture.

The mixture was shaken in a Mixer Mills MM400 mill (Retsch, Germany) for 15 minutes at a frequency of 30 Hz, then centrifuged for 7 minutes at 14,000 g. The aqueous phase was taken, 400 μl of chloroform was added to it, and shaken vigorously for 2 minutes. It was centrifuged as in the previous step, then the aqueous phase was taken and 500 μl of isopropyl alcohol was added to it. It was kept in the refrigerator for ~ 15 minutes, after which it was centrifuged for 7 minutes at 14000 g. The precipitate was washed twice with 70% ethanol and dissolved in deionized water. DNA concentration was determined fluorimetrically using a Quibit 3.0 fluorimeter (Fisher Scientific Heat).

Example 12. Isolation of DNA with 4% sodium lauryl sarcosinate together with 1% SDS and a solution of guanidine chloride.

DNA isolation was carried out according to example 11, characterized in that the concentration of SDS was increased to 2%.

Example 13. Isolation of DNA with 4% sodium lauryl sarcosinate together with 2% SDS and a solution of guanidine chloride.

DNA isolation was carried out according to example 11, characterized in that the concentration of SDS was increased to 2%.

Example 14. Isolation of DNA using 4% sodium lauryl sarcosinate together with 4% SDS and a solution of guanidine chloride.

DNA isolation was carried out according to example 11, characterized in that the concentration of SDS was increased to 4%.

Example 15. Isolation of DNA with 4% sodium lauryl sarcosinate together with 6% SDS and a solution of guanidine chloride.

DNA isolation was carried out according to example 11, characterized in that the concentration of SDS was increased to 6%.

Example 16. Isolation of DNA using 1% sodium lauryl sarcosinate together with 2% SDS and a solution of guanidine chloride.

A sample of 0.2 g of frozen soil was taken into a 2 ml test tube with a screw cap. An amount of a matrix of glass beads was added, approximately equal in volume to the soil, after which 350 μl of a guanidine solution - guanidine HCl 240 mM, sodium phosphate buffer 200 mM, pH 7.0, and 350 μl of a solution of 2% SDS with 1% lauryl sarcosinate were added to each eppendorf sodium - TrisHCl 500 mM, SDS 2% (w / v), sodium lauryl sarcosinate 1% (w / v), pH 7.9, as well as 400 μl of a phenol-chloroform mixture.

The mixture was shaken in a Mixer Mills MM400 mill (Retsch, Germany) for 15 minutes at a frequency of 30 Hz, then centrifuged for 7 minutes at 14,000 g. The aqueous phase was taken, 400 μl of chloroform was added to it, and shaken vigorously for 2 minutes. It was centrifuged as in the previous step, then the aqueous phase was taken and 500 μl of isopropyl alcohol was added to it. It was kept in the refrigerator for ~ 15 minutes, after which it was centrifuged for 7 minutes at 14000 g. The precipitate was washed twice with 70% ethanol and dissolved in deionized water. DNA concentration was determined by fluorimetric method using a Quibit 3.0 fluorimeter (Termo Fisher Scientific).

Example 17. Isolation of DNA using 2% sodium lauryl sarcosinate together with 2% SDS and a solution of guanidine chloride.

DNA isolation was carried out according to example 16, characterized in that the concentration of sodium lauryl sarcosinate was increased to 2%.

Example 18. Isolation of DNA using 6% sodium lauryl sarcosinate together with 2% SDS and a solution of guanidine chloride.

DNA isolation was carried out according to example 16, characterized in that the concentration of sodium lauryl sarcosinate was increased to 6%.

Example 19. Isolation of DNA with 8% sodium lauryl sarcosinate together with 2% SDS and a solution of guanidine chloride.

DNA isolation was carried out according to example 16, characterized in that the concentration of sodium lauryl sarcosinate was increased to 8%.

DNA concentration was recorded using a Qubit 3.0 fluorimeter (Termo Fisher Scientific).

As can be seen from the data shown in table 1, the amount of DNA extracted from a soil sample weighing 0.2 g using a commercial kit NucleoSpin® Soil (MACHEREY-NAGEL) was 580 ng. This is the minimum yield of DNA that was obtained during its isolation by the methods described in examples 1-19 (table 1).

When isolating DNA from the soil using only sodium dodecyl sulfate as a detergent (examples 1-5), the best result was achieved with an SDS concentration of 2%. The DNA yield was 2010 ng / 0.2 g of model soil.

If sodium lauryl sarcosinate was used as the sole detergent (Examples 6-10), the best result was recorded when its concentration was 4%. At this concentration of sodium lauryl sarcosinate, 3030 ng of DNA was isolated from 0.2 g of model soil.

When analyzing the data obtained as a result of the implementation of examples 11-19, the synergistic effect of the action of two detergents at the same time became apparent. With the combined action of sodium dodecyl sulfate and sodium lauryl sarcosinate, the minimum amount of DNA extracted from 0.2 g of model soil was 4830 ng, which is 1.59 times higher than the maximum effect of a single application of the analyzed detergents. The combination of detergent concentrations of 4% sodium lauryl sarcosinate and 2% sodium dodecyl sulfate made it possible to isolate 15300 ng of DNA from 0.2 g of model soil. This is more than 5 times higher than the amount of DNA obtained using any of the detergents individually.

Thus, the data presented in table 1, allowed to conclude that a significant synergistic effect of the combined use of sodium lauryl sarcosinate and sodium dodecyl sulfate. A mixture of these detergents, when DNA was extracted from the soil, more efficiently lysed biological objects in the soil, prevented the sorption of nucleic acids on soil particles and the aggregation of biopolymers.

Table 2 shows data comparing the amount of DNA isolated from 0.2 g of model soil using the prototype method and the proposed method. From the data of table 2 it is seen that when using the declared concentrations of sodium lauryl sarcosinate and sodium dodecyl sulfate, the DNA yield exceeded the amount of DNA isolated using the prototype method from 4.5 to 8.7 times.

The concentrations and ratios of detergents found as a result of the experiments made it possible to obtain a maximum DNA yield of the order of 10,000 ng, which was not achieved by the known analogues and prototype.

Information sources:

1. Kit for the isolation of genomic DNA from soil / FastDNA Spin Kit For Soil, MP Biomedicals. Electronic resource: https://www.dia-m.ru/lab/gomogenizatory-fastprep/acs/17086/ (accessed: 3.08.2018).

2. Genomic DNA from soil. User manual. NucleoSpin® Soil. November 2017 / Rev. 07. http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Genomic%20DNA/UM_gDNASoil.pdf (accessed August 3, 2018).

3. Patent 2650865. Russian Federation, IPC C12N 15/10. A set of reagents for DNA isolation / Viktorov D.A., Nikitin A.G., Pimenov S.V., Toropovsky A.N. - 2016146882, declared. 11/29/2016, publ. 04/17/2018, Bull. No. 11. - S. 3.

4. Bintrim S. B., Donohue T. J., Handelsman J., Roberts G. P., Goodman R. M. Molecular phylogeny of Archaea from soil // Proc. Natl. Acad. Sci. USA - 1997 .-- V. 94 (1). - P. 277-282. https://doi.org/10.1073/pnas.94.1.277

5. Grokhovsky S.L. Specificity of DNA cleavage by ultrasound // Molecular Biology. - 2006. - V. 40 (2). - P. 276-283.

6. Nechipurenko Yu.D., Golovkin M.B., Nechipurenko D.Yu., Ilyicheva I.A., Panchenko L.A., Polozov R.V., Grokhovsky S.L. Characteristic features of DNA cleavage by ultrasound // Journal of Structural Chemistry. - 2009. - T. 50, No. 5. - S. 1045-1052.

7. Sagar K., Singh S., Goutam K.K., Konwar B.K. Assessment of five soil DNA extraction methods and a rapid laboratory-developed method for quality soil DNA extraction for 16S rDNA-based amplification and library construction. // J Microbiol. Methods - 2014. - V. 97. - P. 68-73. doi: 10.1016 / j.mimet.2013.11.008.

8. Devi S.G., Fathima A.A., Radha S., Arunraj R., Curtis W.R., Ramya M. A rapid and economical method for efficient DNA extraction from diverse soils suitable for metagenomic applications // PLoS One. - 2015. - V. 10 (7): e0132441. doi: 10.1371 / journal.pone.0132441.

9. RU 2485178, IPC C12N 15/10, B82B 1/00. publ. 06/20/2013,

10. Isolation of DNA from soil samples: guidelines. Russian Academy of Agricultural Sciences. All-Russian Scientific Research Institute of Agricultural Microbiology. - SPb., 2011 .-- 27 p. - prototype.

11. Sodium lauryl sarcosinate. Electronic resource: https://dic.academic.ru/dic.nsf/ruwiki/1541358 (date of access: 03.08.2018).

12. Sodium lauryl sulfate. Electronic resource: http://moepravo.guru/vozvrat-i-obmen/obsshaya-informatsiya/sostav/laurilsulfat-natriya (accessed: 03.08.2018).

Claims (5)

1. A method for isolating DNA from soil, including homogenizing a soil sample by vibration in the presence of grinding media in a solution containing guanidine HCl, sodium phosphate buffer, TrisHCl, sodium dodecyl sulfate (SDS), a phenol-chloroform mixture, followed by centrifugation and precipitation of DNA with isopropanol characterized in that the solution for soil homogenization additionally contains sodium lauryl sarcosinate detergent in the following ratio of starting components, wt.%: sodium lauryl sarcosinate - 2-6, sodium dodecyl sulfate - 1-4. 2. The method according to p. 1, characterized in that the homogenization of the soil sample is carried out using a vibrating mill in the frequency range 25-35 Hz. 3. The method according to p. 1, characterized in that as the grinding media use a mixture of glass beads with a diameter of 0.5 mm and 1.0 mm and ceramic beads with a diameter of 2.0 mm in a weight ratio of 3: 1: 1, respectively.