Method for qualitative and quantitative analysis of lipids strongly bound with genomic dna

FIELD: chemistry.

SUBSTANCE: invention relates to molecular biology and genetics. Disclosed is a method for qualitative and quantitative analysis of lipids that are strongly bound with genomic DNA, which comprises steps of isolating genomic DNA bound with lipids from cells using a detergent method, hydrolysis of DNA with a hydrolysing enzyme, separating lipids from the chloroform-methanol-water mixture at 37°C, evaporating the solvent, stabilising lipids with 2,6-di-tert-butyl-p-cresol, mixing the lipids with TMSH, chromatography with a gas chromatograph, followed by mass spectrometry.

EFFECT: method can be used to decode the lipid code of genomic DNA, ie, to determine the area of localisation of lipids that are strongly bound with genomic DNA for research and therapeutic purposes.

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The claimed technical solution relates to the field of molecular biology and genetics and can be used to determine the fatty acid composition of lipid fractions, strongly bound with DNA. This result is the most important factor decryption code of lipid genomic DNA, i.e. to determine the sites of localization tightly bound lipids in the genomic DNA.

Currently known a number of biochemical methods for the isolation of natural supramolecular complexes of DNA-lipids (hereinafter ncddc) from various sources, namely:

- phenol method using extraction of impurities with a saturated aqueous solution of phenol (Struchkov et al., 2002),

- detergent method of allocating fractions of lipids tightly bound to DNA, without the use of phenol (Zhdanov et al., 2006, FEMS Environ. Lett.).

About the properties of these supramolecular complexes genomic DNA is known that DNA in them is characterized by a high molecular weight, and they are at least 4 types of biomacromolecule: RNA (10-15 weight%), protein (1-3%), lipids (1-3%), the rest is a DNA (Pods, Struzewska, 2000). The composition of the protein components of this complex may include three or four small DNA-binding protein by weight 40-60 kDa (Pods et al., 1990). Compared with detergent, phenol method has a significant disadvantage in prob is gnosti solubilization of membrane lipids in the fraction of DNA and their exchange with the fraction of DNA-bound lipids. Detergent method can lead to a decrease in the output of the supramolecular complex DNA. About lipid component ncddc known only to the fatty acid composition of the two fractions of DNA-bound lipids: soluble and strongly bound (Zhdanov et al., 2006 a,b).

About the properties and functioning of such multicomponent biomacromolecular complexes in the cell, very little is known, though they are certainly important for the functioning of organisms. The main problems are: clarification of the nature of interaction of lipids with DNA and relationships of four types of biomacromolecule - lipids, DNA, RNA and proteins.

Known technical solution according to the patent of Russian Federation №2439580, the method for determining the fractions of lipoproteins in the blood of patients with ischemic heart disease by electrophoresis in agarose gel by treating the serum samples of the patient's blood within 1 h with a solution of the dye Sudan B in a dark thermostat at 40°C, the heated mixture is introduced into the well of the agarose gel with a volume of 4×20×10 mm with subsequent fixation of electrophoregram, their drying and densitometry, characterized in that it further before processing Sudan B to the 0.3 ml sample of blood serum of the patient add 0.1 ml of 0.1%solution of Triton X-100 and incubate 15 min at 20°C, stirred the mixture with the shaking method 120 times in 1 min and hold the disintegration of lipoproteins.

Yes what a great invention relates to the field of medical biochemistry, in the work were extracted and separated into fractions of cellular lipoprotein in patients with coronary heart disease. The authors of the above-described invention does not aim was to investigate the interaction of the lipids and lipoproteins of cells with genomic DNA or oligo - or polynucleotide DNA, so in the famous invention does not provide information about the interaction of the components of lipidome and lipoproteins with DNA.

Subject to the claimed technical solution is the investigation of the nature of the interaction of the complexes of lipids and DNA.

We have identified the fraction of lipids associated with prokaryotic DNA, namely the fraction of weakly and strongly bound fraction with DNA, lipids, and investigated the fatty acid composition of strongly bound fractions of lipids.

These properties and the incompleteness of the description above, the invention according to patent No. 2439580 due to the fact that the technical solution for the implementation of diagnosing diseases based on the analysis of the composition of cellular lipids. It is important to note that the main method was chromatographic analysis, as in the claimed technical solution used in the analysis of fatty acid composition of lipids by gas chromatograph and mass spectrometry.

Of the investigated prior art, the applicant identified the technical solution according to the patent of Russian Federation №2062468, extraction and separation is gunning lipids by treatment of the cells with a mixture of non-polar solvent and a polar alcohol followed by thin-layer chromatography, characterized in that to increase the efficiency and accuracy of determination of intracellular lipids from whole cells pre-applied on the coated silica gel plates and then carried out the extraction and separation of lipids in the ascending current of solvent of the following composition: chloroform: methanol: 7M ammonia: water in the ratio of 65:25:1:3, respectively.

Known technical solution relates to the field of biochemistry and in the opinion of the applicant, is closest to the claimed technical essence and the achieved result. However, according to the applicant, it cannot be selected as the closest analogue due to the fact that there the authors carried out the extraction and separation of cellular lipids, and in our work we determined the fatty acid composition of DNA-bound lipids.

The authors of the famous above-described technical solutions do not aim was to investigate the interaction of the lipids and lipoproteins of cells with genomic DNA or oligo - or polynucleotide DNA, and therefore their description of the patent specification does not provide information on the interaction of lipids and lipoproteins with deoxyribonucleic acid. As in the previous patent, the claimed technical solution to the main research method was chromatographic analysis.

Based on the foregoing conclusions can be drawn about from outstay analogues of the claimed technical solution, as identified from known at the filing date of the prior art technical solutions are not designed to resolve their goals and not allow challenges for qualitative and quantitative analysis of lipids tightly bound to the genomic DNA.

The applicant claimed technical solution is proposed biochemical approach to answering this question, namely the processing of the drug ncddc isolated from Pseudomonas aurantiaca, in parallel with different enzymes, gidrolizuemye or DNA, or RNA, or proteins (proteinase K).

Analyzing changes in fatty acid composition of the complex after treatment with enzymes, the applicant draws conclusions about which of the treatments resulted in different changes in fatty acid composition and which model we are inclined in the analysis and identification of the structure of the supramolecular complex of DNA.

In the claimed technical solution is shown, in particular, that the main components of fatty acid composition of the DNA-bound lipids of the preparation of genomic DNA extracted detergent method, are acid 16:0, 18:0 and 10:0, 11:0 2OH, 13:1 and 13:0 2OH acid contained in a smaller number (Figure 1). The data presented in Figure 1-4 shows that hydroxyacids 11:02OH and 13:02OH are part of the lipid-related proteins; 14:1ω5c - related proteins and RNA, and 15:1ω8c - related DNA.

Research the research in this direction, according to the applicant, was not conducted in any of the University or academic laboratories in the world.

The results can then be used in the development of new methods of diagnostics of diseases of lipid DNA code of the patient or in the development of new types of drugs. Thus, the purpose of the claimed technical solution is to develop a method of analysis of the structure of the supramolecular complex DNA by treatment with various gidrolizuemye enzymes with subsequent fatty acid analysis of remaining tightly bound to DNA, lipids.

The essence of the claimed technical solution is that the way quantitative and qualitative analysis of lipids tightly bound to the genomic DNA, involves the following stages:

- selection of cells genomic DNA associated with lipids, detergent method;

- hydrolysis of DNA gidrolizuut enzyme;

- selection of lipids in a mixture of chloroform-methanol-water at 37°C;

evaporation of the solvent;

- stabilization of lipids 2,6-di-tert-butyl-p-cresol;

- mixing lipids with TMSH;

- chromatography using a gas chromatograph with subsequent mass spectrometry.

The claimed technical solution is illustrated by the drawings, figures 1 to 5 and examples.

Figure 1 shows how the quality and quantity of the aqueous analysis of lipids, tightly bound with genomic DNA (content of fatty acids in raw enzymes sample of DNA complexes with proteins, RNA and lipids).

The abscissa axis fatty acids, on the y - axis the content of fatty acids (%) numbers in order: 1 - 10:0, 2 - 11:0 2OH, 3 - 13:1 (12, 13), 4 - 13:0 2OH, 5 - 16:0,6 - 18:0.

Figure 2 shows how the qualitative and quantitative analysis of lipids tightly bound to the genomic DNA (content of fatty acids in the treated enzyme Dnazol sample of DNA complexes with proteins, RNA and lipids).

The abscissa axis fatty acids, on the y - axis the content of fatty acids (%) numbers in order: 1 - 10:0, 2 - 11:0 2OH, 3 - 14:0, 4 - 14:1ω5c, 5 - 16:0, 6 - 18:0.

Figure 3 shows how the qualitative and quantitative analysis of lipids tightly bound to the genomic DNA (content of fatty acids in processed by the enzyme RNase sample of DNA complexes with proteins, RNA and lipids).

The abscissa axis fatty acids, on the y - axis the content of fatty acids (%) numbers in order: 1 - 10:0, 2 - 13:0 2OH, 3 - 14:0, 4 - 15:1ω8c, 5 - 16:0, 6 - 18:0.

Figure 4 shows how the qualitative and quantitative analysis of lipids tightly bound to the genomic DNA (content of fatty acids in processed by the enzyme protease To the sample of DNA complexes with proteins, RNA and lipids).

The abscissa axis fatty acids, on the y - axis the content of fatty acids is (in %), rooms in order: 1 - 10:0, 2 - 14:0, 3 - 15:1ω8c, 4 - 16:0, 5 - 18:0, 6 - 18:3ω6c (6,9,12).

Figure 5 shows how the qualitative and quantitative analysis of lipids tightly bound to the genomic DNA (block-scheme for strongly and weakly bound to DNA, lipids).

Example

The claimed technical solution is illustrated in the following example, which is not exhaustive, but gives an idea of the possibilities of practical implementation of the claimed technical solution.

The essence of implementing the claimed solution is the following: the same samples supramolecular complex genomic DNA of the cells undergo hydrolysis of three different enzymes with subsequent dialysis:

or Dnazol,

or RNase,

or proteinase.

From the samples extracted lipids that are analyzed by gas chromatography to obtain the fatty acid profiles (see Figure 5).

The results of fatty acid markers (biomarkers) DNA-bound lipids that enables the development of new medicines type.

The claimed technical solution provides the ability to determine the fatty acid composition of the lipid fraction, strongly bound with DNA, which remains after processing ncddc different gidrolizuemye enzymes for drug development new t the PA.

To confirm the identification and analysis of lipids tightly bound to the genomic DNA, conducted the experiments described in the example below, based on the fact that the tightly bound lipids are not soluble in detergent and alcohol unlike reprocessing that sportgastein.

Identification and analysis of lipids tightly bound to the genomic DNA was carried out as follows (Figure 5).

Genomic DNA extracted from cells by the method based on the use of detergents.

Cells lyse (destruction of the cell membrane) detergent-salt method to treatment with lysozyme in 20% sucrose.

Cells are precipitated by centrifugation at 3000 rpm for 5 min and resuspended in 9.5 ml of 50 mm Tris-HCl, pH=8.0, 50 mm NaCl, 5 mm EDTA.

- Then, to the suspension is added 1.5 ml of a solution of lysozyme (50 mg/ml and incubated for one hour at 37°C.

- Then, to the suspension is added 1.5 ml of 10% solution of sodium dodecyl sulfate for processing cells according to the method described by Cunha et al., 2001.

Then the fraction soluble in ethanol lipids extracted 35% aqueous solution of ethanol at 37°C for 24 hours. DNA is precipitated with ethanol and the precipitate washed twice with 70% aqueous solution of ethanol.

Obtained extracts are combined and then dried under reduced pressure to obtain a soluble fraction DNA bound the data lipids.

- The remaining sludge is prepared and processed depending on gidrolizuemye enzyme as follows:

or for Gnkazy I:

The resulting residue was dissolved in 5 μl of buffer solution (20 mm Tris-Hcl (pH 8,4), 2 mm MgCl2, 50 mm KCl) and processed by 1 unit Gnkazy I in 5 μl of 10 mm p-RA of magnesium chloride. After incubation for 2 hours Tnkase I heat-inactivated for 20 minutes at 95°C.

or for RNase A:

The resulting residue is dissolved in 20 µl of assay buffer RNase A (10 mm Tris-Hcl, 15 mm NaCl, pH 7.5) and thereto is added 1 μl of a solution of 1 unit/μl RNase A. After two-hour incubation at 37°C is thermal inactivation of RNase for 5 min at 95°C.

or for the proteinase K:

The residue is dissolved in 20 µl of 6 M guanidine-hydrochloride, 50 mm Tris-hydrochloride, 5 mm DTT, pH=7,5. The solution was heated at 95°C, 20 minutes After denaturation of proteins, the mixture is cooled to room temperature. Added 50 μl of 50 mm Tris-Hcl pH level of 7.5. Then added 20 μg of proteinase K and incubated 90 min at 37°C. For inhibition of the action of proteases samples are placed in a boiling water bath for 20 minutes

After two hours incubation of the mixture at 37°C tightly bound to DNA, lipids are allocated using a mixture of chloroform-methanol-water according to the method described by Bligh&Dyer, 1959. The solvent is removed by evaporation at Pont the leaders introduce pressure. To prevent aging samples lipids are stabilized with 2,6-di-tert-butyl-p-cresol and stored under nitrogen atmosphere in kelvinator at -80°C. Fatty acids in all samples analyzed in the form of their derivatives of methyl esters using gas chromatograph connected to a mass spectrometer. After adding TMSH to sample all traces of lipids fatty acids are converted into Quaternary sulfonate salt, which pyrolized to methyl esters in the hot injection into the gas chromatograph.

Loss of short-chain fatty acids are avoided by transesterification taking place at room temperature, and no extraction step after chemical modification of fatty acids.

Chromatographic separation is performed using capillary column made of quartz glass, as described previously.

The proposal demonstrates that the main components of fatty acid composition of the DNA-bound lipids of the preparation of genomic DNA extracted detergent method (Figure 5), are acids such as 16:0, 18:0 and 10:0, 11:0 20N, 13:1 and 13:0 2HE acid contained in a smaller number (Figure 1). The data presented in Figure 1-4 shows that the hydroxy acids - 11:ON and 13:ON are part of the lipid-related proteins; 14:1ω5 - related proteins and RNA, and 15:1ω8 - related DNA.

The claimed technical solution provides the ability op is adelite fatty acid composition of the lipid fraction, tightly bound to DNA, which remains after processing ncddc different gidrolizuemye enzymes for drug development new type

To realize these objectives previously was not possible, as it was not developed method of determination of fatty acid composition of the lipids associated with the components of the chromosome: either DNA or RNA, or proteins.

Based on the above, the claimed method provides the opportunity to achieve the stated goals - provides the ability to determine the fatty acid composition of the lipid fraction remaining after processing ncddc different gidrolizuemye enzymes that will be important for drug development of the new type.

Implementing the determination of fatty acid composition of the lipid fraction remaining after processing ncddc different gidrolizuemye enzymes, ensures the development of new methods of diagnostics of diseases of lipid DNA code of the patient or can be applied in the development of new types of drugs. Thus, the claimed technical solution provides for the development of method of analysis of the structure of the supramolecular complex DNA by treatment with various gidrolizuemye enzymes with subsequent fatty acid analysis of remaining tightly bound to DNA, lipids.

The way the number is i.i.d. and qualitative analysis of lipids, tightly bound with genomic DNA, including the stage of discharge cells genomic DNA associated with lipids, detergent method, DNA hydrolysis gidrolizuut enzyme, separation of lipids in a mixture of chloroform-methanol-water at C, evaporation of the solvent stabilization of lipids 2,6-di-tert-butyl-p-cresol, mixing lipids with TMSH, chromatography using a gas chromatograph with subsequent mass spectrometry.



 

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