A method of obtaining nucleic acids from bacteria

 

(57) Abstract:

Usage: biotechnology, Microbiology. The essence of the invention: obtaining nucleic acid from biomass of bacteria, for this purpose, the biomass of bacteria Methylococcus capsulatus, which is subjected to extraction with water or alkaline water-salt solution at a pH not above 7.5. The first stage is separated ribonucleic acid (temperature 85-90oC) on the second stage at a pH of 8.6-9.0 to separate a mixture of DNA and RNA, which are then separated by known methods. The method allows to obtain preparations of DNA and RNA of high purity.

The invention relates to Microbiology and biotechnology, and in particular to methods of producing nucleic acids and can be used in food, chemical and medical industries.

The method for extracting nucleic acids from Bogoroditsa bacteria type Alcaligenes eutrophus Z-1, namely, that the suspension of cells of bacteria Alcaligenes eutrophus Z-1 is treated with a mixture of benzyl or isobutyl alcohol (20-30:5-30% by volume), or benzyl alcohol, and chloroform (30-40: 10-20% by volume), mix, leave at room temperature for 40 min 4 h, then diluted with 3-10 times 5% NaCl and 0.4% Na-citrate, Zentai, but it does not provide an integrated use of raw materials, because the bacteria after treatment in heavily contaminated with organic substances, which makes it impossible for their future use. The second disadvantage of this method is that the final product is a mixture of nucleic acids for separation which require additional costs.

The task of the invention is to develop a method of obtaining nucleic acids without the use of organic substances from bacterial biomass, which allows to obtain pure ribonucleic acid (RNA) and a mixture of deoxyribonucleic acid (DNA) and RNA.

The problem is solved in that in the method of obtaining nucleic acids from bacteria as bacteria use biomass of native cells metanalysis bacteria Methylococcus capsulatus, and the process of extraction of nucleic acids conduct water-base or water-salt solutions in series, the first stage separated RNA at a pH of not greater than 7.5 and t=85-90oC, in the second stage, separating a mixture of RNA and DNA at pH=8,6-9.0 and t=85-90oC division by known methods.

For the first stage of preparing an aqueous-alkaline WM and water-salt suspension ammonium salts of orthophosphoric acid with a concentration of phosphate of 1.5 to 1.75 mol/l, pH7,5, the suspension is heated at t= 85-90oC for 1 h, separated extract RNA from the biomass by centrifugation (10 min, 6000 rpm). From the obtained extract isolated RNA preparation by known methods with the content of the basic substance > 80%

In the second stage to separate a mixture of DNA and RNA from the obtained after centrifugation of biomass, for which the obtained residue biomass plant water-base or water-salt solutions with pH8,6-9,0 and heating the suspension at 85-90oC for 2 hours Denuclearizing biomass is separated from the extract by centrifugation (10 min, 6000 rpm). From the extract by known methods produce purified mixture of DNA and RNA with DNA content>50% and RNA>30%

The invention is illustrated by the following examples:

Example 1.

To 2040 g biomass metanalysis bacteria Methylococcus capsulatus with DM contents of 10% and a content of nucleic acids 8.7% in terms of dry substance, including RNA 7.3% and DNA 1,2% poured 3000 ml of water-salt solution of ammonium salt of phosphoric acid with phosphate content of 3.3 mol/l, pH7,5, mix. The resulting suspension is heated to 90oC, kept at this temperature, with moderate stirring, for 1 h At the end of the process extricate 3865 ml of extract containing nucleic acid 2.5 g/l and 1267 condensed biomass of bacteria. From the resulting extract by known methods get 3,82 g product RNA with the following characteristics: sod.total NK 80% of the DNA of 1.03% protein 4,2% moisture 10%

The release of RNA from the total na content in the cells is 21%

To 1267 obtained water-salt sediment bacterial biomass type 3400 ml ammonium salts of orthophosphoric acid with the phosphate content of 1.75 mol/l and a pH of 8.6, mix. The obtained water-salt suspension is heated to 90oC and kept at this temperature and under moderate stirring for 2 hours After the end of the process, the suspension is cooled to room temperature and centrifuged. Get 3350 ml of water-salt extract containing NC 1.3 g/l Of the obtained extract by known methods allocate 400 mg DNA concentrate with the following characteristics: sod. total NK 80% of the DNA is 50% protein and 4.5% moisture 10%

The output DNA concentrate on the total na content in the cells is 1.5%

Example 2

To 3140 g biomass metanalysis bacteria Methylococcus capsulatus with DM contents of 20% and a content of nucleic acids calculated on the dry substance of 8.5% including RNA 7.2% and DNA 1,3% poured 5020 ml with distilled water and mix. Obtained a suspension of the title is within 1 hour At the end of the extraction process, the suspension is cooled to room temperature and remove the bacterial biomass by centrifugation. Get 4750 ml of extract containing NC 2.2 g/l and 3400 g condensed biomass of bacteria. From the resulting extract by known methods get 3 g of purified RNA preparation with the following characteristics: sod.total NK 80% DNA 3.5% protein 2,07% moisture 10%

The release of RNA from the total na content in the cells is 11.5%

To 3400 g the sediment bacterial biomass with DM contents of 20% add 9900 ml of distilled water and mix. The resulting suspension is titrated ammonia water to pH of 9.0, heated to 85oC and under stirring withstand this suspension 2 hours After the end of the process, the suspension is cooled to room temperature and centrifuged. Get 9820 ml extract concentration NC of 1.3 g/l Of the obtained extract by known methods allocate 340 mg DNA concentrate with the following characteristics: the content of total NK 80% of the DNA is 50% protein, 4.8% moisture 10%

The output DNA concentrate on the total na content in the cells is 1%

As seen from the above examples, the above-described method of obtaining NC bacteria Methylococcus capsulatus allows wheat 5 and DNA concentrate containing DNA more than 50 and admixture RNA is not above 30 protein of not more than 5% This method eliminates the use of organic substances in the extraction step as RNA, so DNA, which prevents contamination of the biomass of undesirable impurities and allows its use in further processing.

A method of obtaining nucleic acids from bacteria, characterized in that the biomass of cells metanalysis bacteria Methylococcus capsulatus subjected to extraction with water or alkaline water-salt solution and the rst allocate RNA at pH 7.0 to 7.5 and a temperature of 85 90oAnd then at pH 8,6 9,0 and the same temperature emit a mixture of DNA and RNA and their subsequent separation.

 

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