SU1661217A1 - Method for nucleic acids isolation from hydrogen-oxidazing bacteria alcaligenes eutrophus z-1 - Google Patents

Abstract

This invention relates to applied biochemistry and molecular biology, in particular to methods for producing nucleic acids. The aim of the invention is to increase the yield of nucleic acids, to simplify, speed up and reduce the cost of the isolation procedure. A distinctive feature of the method is the reduction in the number of stages in the process of isolation — cell disruption, extraction and deproteinization are carried out simultaneously. Namely, the suspension of bacterial cells ALCALIGENES EUTROPHUS Z = 1 is treated with a mixture of benzyl and isobutyl (20 - 30: 5 - 30% by volume) or benzyl alcohol and chloroform (30 - 40: 10 - 20% by volume), mixed, left at room temperature for 40 minutes - 4 hours, then diluted 3 to 10 times with 5% NACL and 0.4% NA-citrate, centrifuged, and nucleic acids from the supernatant precipitated with standard. The yield of the drug is 65 - 70%, the protein content is insignificant. The method can be used in large-scale production, in addition, in the practice of laboratory work on biochemistry and molecular biology. 1 tab.

Description

The invention relates to the field of applied biochemistry and molecular biology, in particular, to methods for the preparation of nucleic acid preparations.

The purpose of the invention is to simplify the method and increase the yield of the target product.

The destruction of bacterial cells, the deproteinization and extraction of the final product is carried out in one stage. Suspension of biomass of 3–16% calculated on the dry weight in a solution of 5–6% NaCI and 0.4–0.5% Na-citrate is mixed with two types of systems: benzyl and isobutyl alcohols (final concentration 20 -40 and 5-30 vol.%, Respectively) or benzyl alcohol and chloroform (final concentration 20-40 and 10-20 vol.%

respectively) for 40-360 minutes until a stable emulsion is formed, from which nucleic acids are extracted with a solution of 5-6% NaCl and 0.4 M Na-citrate. Debris and denatured proteins are removed by centrifugation (10 min, 4000 rpm), and nucleic acids from the supernatant are precipitated by adding ethanol (65-70 vol.%) At 0-4 ° C, 4 h. All operations are carried out at room temperature.

The method provides 65-70% yield of nucleic acids from Alcaligenes eutrophus Z-1 cells, the resulting preparations are practically free from protein impurities.

Example 1.3 g of biomass containing 14.7% of dry matter and 11.2% of nucleineo

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acid, mixed with 1.2 ml of a saturated solution of NaCl in 2.4% citrate and 4.2 ml of water. A 6% (on a dry basis) suspension in 5% NaCI and 0.4% Na citrate is obtained, mixed with 2.1 ml of benzyl and 1 ml of isobutyl alcohol, which corresponds to 20 and 10% by volume. The emulsion was kept for 120 minutes, diluted 10-fold with 94 ml of 5% NaCl and 0.4% Na citrate, separated by centrifugation and discarded. Nucleic acids are precipitated from the solution with two volumes of ethanol at 0 ° C, and the precipitate is collected by centrifugation for 10 minutes at 4000 rpm. The precipitate contains 39.6 mg of nucleic acids, the precipitate does not contain protein. The yield of nucleic acids 68%.

Example 2. A charge of biomass containing 12.9% dry matter and 10.0% nucleic acids, calculated on the dry weight, is mixed with 1 ml of a saturated solution of NaCl in 2.4% Na citrate. A 10% suspension of biomass is mixed with 2.4 ml of benzyl alcohol and 1.6 ml of chloroform, which corresponds to 30 and 20% by volume, respectively. The emulsion is incubated for 180 minutes at room temperature, diluted 10 times with 72 ml of 5% NaCl and 0.4% Na citrate, separated by centrifugation at 4000 rpm for 10 min and the supernatant is precipitated and the nucleic acids are precipitated with two volumes ethanol at 0 ° C. The precipitate is collected by centrifugation at 0 ° C, 4000 rpm. The precipitate does not contain protein, contains 27.1 mg of nucleic acids, which corresponds to a 70% yield.

In tab. 1 and 2 show the results of nucleic acid production.

The proposed method allows one to achieve a more complete yield of nucleic acids from Alcallgenes etrophus Z-1 cells (65-70%) as compared to the isolation results by known methods (up to 45%). The low content of protein impurities in the resulting nucleic acid preparation is also important. The method differs from the known simplicity and short duration, as well as the reduction of the deproteinization time with toxic substances (chloroform, phenol). In addition, the cost of the final product is reduced by the use of affordable low-cost reagents.

The proposed method can be used in the large-scale production of nucleic acids - an important product that is the basis for obtaining a number of the most valuable pharmacological

drugs. In addition, the proposed method can find application in the practice of molecules rnobiological and biochemical studies.

Claims (1)

Invention Formula Nucleic Acid Extraction Method from hydrogen-acid bacteriaA1caIdepe8 lysis eutrophus Z-1 cell separation of nucleic acid solution acids from cell debris, its deproteinization and nucleic acid precipitation with alcohol, characterized in that, in order to simplify the method and increase the yield of the target product, lysis of cells with simultaneous deproteinization is carried out by adding 3-16% cell suspension to 5-6% NaCl and 0.4-0.5% Na citrate mixture 20-30% vol. benzyl alcohol and 5-30% vol. isobutyl alcohol or mixture 30-40% by volume of benzyl alcohol and 10-20% by volume of chloroform, stirring, keeping at room temperature 40- 240, min. Diluting the emulsion 3-10 times with a mixture of solutions of 5-6% NaCI and 0.4-0.5 % Na-citrate and supernatant separation from proteins and cell derbies, and nucleic acid precipitation is carried out with 65-70% by volume ethanol. Table 1 table 2