Method of marking human 7th chromosome in metaphasic and interphasic cells

FIELD: medicine.

SUBSTANCE: what is offered is a method of marking a human 7th chromosome providing in situ hybridisation of metaphasic or interphasic cell chromosomes of a tested sample and a DNA probe presented by a marker plasmid alpha R1-13 which consists of EcoR 1-EcoR l DNA fragment of a pBR 325 vector of the value 5966 base pairs and EcoRl-EcoRl alphoid DNA fragment of the human 7th chromosome of the value 680 base pairs. The testing environment in which the used DNA-probe specifically interacts with a centromeric region of the 7th chromosome without cross hybridisation with other human chromosomes are developed.

EFFECT: more efficient identification of the presented chromosome and enabled application of the new method in medical diagnostics.

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The invention relates to medical genetics, in particular to the field of genetic counseling and laboratory diagnosis of chromosomal diseases, and can be used for reliable identification of the 7th chromosome in norm and pathology, including preimplantation, prenatal and postnatal diagnosis of various forms of structural chromosomal abnormalities involving 7 of chromosome aneuploidy, ring and additional marker chromosomes using cultivated or non-cultivated human cells.

The aim of the invention is to develop an effective way of marking the 7th human chromosome in metaphase and interphase cells using the original molecular genetic marker specific for the centromeric region of the 7th chromosome, including the heterochromatin of both shoulders, which has the following advantages to accelerate and improve the accuracy of the method is:

1. When applying specific conditions in situ hybridization and post-hybridization washing cytological preparations strictly marked allocentrically district 7 of chromosome without cross-hybridization to other chromosomes.

2. Effectively marked the centromeric region 7-th chromosome in interphase nuclei to identify numerical chromosomal aberrations that n is necessary for karyological analysis of non-dividing cells, for example cell tumors.

The essence of the invention lies in the fact that the original cloned fragment altenau DNA marking the centromeric region 7 of chromosome person, used as DNA probes for differential identification of the 7th chromosome specific conditions for in situ hybridization, which allows you to quickly and reliably determine the number of chromosome 7 in interphase nuclei or metaphase human cells.

It is known that by now cloned and characterized DNA probes for different human chromosomes. However, as DNA probes often use unique (odnokorennye or melakopides DNA sequences)that do not allow efficient molecular cytogenetic diagnosis due to the low resolution of modern cytogenetic techniques. The exception is chromosomespecifc DNA probes containing repetitive (mnogoopytnyi) DNA sequences. These include satellite (alpha and "classical" satellites) DNA that are repeated from several hundred to several thousand times relatively short (up to 170 nucleotides) DNA fragments, forming the centromeric regions of all human chromosomes. On their basis, created and protected by copyright certificates DNA probes and haigney DNA with specificity for chromosomes 1, 3, 4, 6, 9, 11, 13, 18, 21 and X person (ghindilis and others, 1985, No. 1203108; Yurov and others, 1989, No. 1494724; Yurov and others, 1993, No. 1792429; Yurov and others, 1997, No. 2087533; Soloviev and others, 1997, No. 2087534, Yurov and others, 2000, No. 2161199; Yurov and others, 2005, No. 2265060, Yurov and others, 2008, No. 2325441). The challenge remained to obtain DNA probes for efficient labeling of other human chromosomes. Currently describes the number of cloned alpha-satellite and classical satellite DNA, which are characterized by specificity for different human chromosomes. These sequences can be considered as a prototype proposed for use in this invention recombinant plasmids. However, the possibility of their use for marking and identification of human chromosomes remains open. In particular, the detected variants altenau human DNA, which are available in 7-th chromosome (Product catalogue Vysis FISH probes "Abbott Molecular Diagnostics", www.abbottmolecular.com). These DNA probes are not investigated in sufficient detail with regard to their design for marking the 7th chromosome person in applied work, in particular, for the diagnosis of chromosomal abnormalities in Oncology and medical genetics. In this case, all described in the literature, the methods of affixing the 7th chromosomes in metaphase and interphase cells and the use of recombinant plasmids containing alpha-satellite DNA, do not have sufficiently high is some specificity for the centromeric regions of chromosomes 7, and, therefore, are intended for laboratory research only, not for diagnosis of chromosomal abnormalities when carrying out genetic counselling. Therefore, they cannot be used for effective molecular cytogenetic diagnosis in medical practice when you want the correct identification of abnormalities of chromosomes and subsequent genetic counselling and prenatal diagnosis with possible termination of pregnancy due to aberrations of the 7th chromosome. Relevant for molecular cytogenetic diagnostics is to develop effective ways of identifying chromosomes and design of the original markers for the centromeric region of the 7th chromosome of man, which would allow for the identification of centromeric regions in interphase and metaphase cells.

This method for highly specific labeling centromeric regions 7 of chromosome person using the original recombinant plasmids alpha R1-13 developed for the first time. The obtained recombinant plasmid R1 alpha-13 differs from the known in literature cloned alpha-satellite DNA on a set of attributes: the method of obtaining and high specificity for the 7th chromosomes of man. Special conditions for in situ hybridization using standard with the left solution with 55% formamide for 4-18 hours at a temperature of 42°C and postliberalization washing in saline solution with 50% formamide at a temperature of 42°C. this DNA probe hybridized only with the centromeric regions of the 7th chromosomes of man. The proposed method of affixing the 7th chromosome for chromosome pathology diagnosis does not have the above common features with any of the above equivalents, as for molecular cytogenetic labeling and identification of centromeric regions 7 of chromosome man this technical solution is developed for the first time.

The specific objective of the study was achieved as follows. Source selection marker fragment alpha R1-13 is the DNA of human lymphocytes, purified by the method of electrofiltration on ultrafiltrable membrane type HM-300 "Amicon". The preparation of purified lymphocytic human DNA (obtained from individual male) treated with restriction enzyme EcoRl to complete hydrolysis. Received restrictive fragments, the size of 680 base pairs preparative allocate by electrophoresis in 0.8% of agarose gel and "sewn" by ligating the "sticky" ends in the EcoRl site of the bacterial plasmid vector pBR325 (5966 pairs of nucleotides). This plasmid carries the gene for resistance to ampicillin. The presence of a resistance gene for ampicillin plasmid allows on the environment with this antibiotic to select transformed cells. Thus, this vector allows you to create a recombinant multicopy (like "short-gun") with automatic selection on medium containing ampici the Lin.

The next step in the multicopy identify clones having homology with altenau DNA. For this recombinant clones on nitrocellulose filters is introduced into the reaction hybridization with labeled with radioactive phosphorus (32P) total DNA fraction ARI-human DNA, which is represented mainly altenau DNA. Colonies, DNA which give positive reflections on radioautographic, by hybridization of selected and used to determine the chromosomal localization of the cloned them restricted fragments of human DNA, directly in cytological preparations of chromosomes in situ.

For this chromosome is made from dividing cells in culture of peripheral blood lymphocytes by a known method. Stimulated to divide with the help of phytohemagglutinin (firm "Difco, USA) blood lymphocytes cultured in penicillin vials at 37°C in medium Needle with the addition of up to 20% bovine serum for 72 hours. 1.5 hours before the end of the cultivation injected colchicine at a concentration of 0.5 μg/ml Cells in the environment of the cultivation transferred into centrifuge tubes and centrifuged 5 min at 1000 rpm Sediment resuspended in hypotonic solution of 0.07 M KCl, and incubated 10 min at 37°C. Fixation spend methanol-acetic acid fixative in the ratio of 3: 1 three times for 20 minutes P is aparaty chromosomes stored at a temperature of 37°C and used in the experiments no later than 2-3 weeks after preparation.

DNA samples for hybridization on chromosomes in situ mark isotopic label known method of substitution; in 100 μl of deionized water dissolve consistently 15 ál of ten buffer solution (0.8 M Tris-HCl, pH 7.4; 0.1 M MgCl2; 0,05 M dithiothreitol), 5 ál of non-radioactive deoxynucleotides in equimolar mixture of (0.01 mmol of each except timeinterest), 5 μl of dnaase-1 (concentration 1 μg/ml), 10 ál3N-timeinterest (specific activity 114 CI/mol, concentration of 5 MCI/ml, 10 ál of DNA-polymerase-1 at a concentration of 2 u/μl). The volume of the reaction mixture is 150 μl. The reaction is carried out 20-40 minutes at a temperature of 20°C. the Average specific radioactivity of labeled DNA preparations is about 25×10 pulses per min per 1 ág DNA.

The cloned fragments altenau DNA were mapped on human chromosomes in situ using standard conditions is hybridization in a solution containing a standard saline solution (2×SSC), 50% of formamide, 10% textresult-500. All clones were hybridisable with the centromeric regions of all human chromosomes with no apparent specificity for individual chromosomes. However, the application of special conditions of hybridization and high stringency hybridization by increasing the concentration of formamide in solution up to 55% helped identify recombinant DNA al is and R1-13, which is fundamentally different on the chromosome specificity from other altenach DNA.

Hybridization of labeled radioactive precursor recombinant DNA alpha R1-13 with metaphase chromosomes in situ carried out by the following procedure: preparations of metaphase chromosomes denature for 30 seconds in a 0.07 N NaOH, followed by washing for 10 minutes in solutions of 70° and 96.6° alcohol; drugs radioactive DNA samples are denatured in hybridization mixture containing 55% solution of formamide, 10% solution of dextransucrase-500 and a standard saline solution (2×SSC)for 10 min at a temperature of 100°C. Hybridization was performed at 37°C for 17-18 hours. Drugs after conducting hybridization washed in two changes of 2×SSC at room temperature in solutions of 70° and 96.6° ethanol for 15 min in each shift. After drying in air the drugs covered by the emulsion type M and exhibit in the dark for 10 days. After developing, the standard apidology a developer of drugs for 2 min, washed thoroughly with running water and paint 3%dye solution Romanovsky-Gimza in 0.02 M phosphate buffer (pH 6.8) for 10-15 min Radioautography analyzed and photographed under a microscope at magnification × 1000, × 1125.

Samples of recombinant DNA alpha R1-13 for hybridization on chromosomes in situ is you can tag and non-radioactive fluorescent nucleotides, for example Biotin-16-dUTP (Biotin-16-deoxy-uridinediphosphate), the above-described method of substitution. To do this, prepare the reaction mixture of the following composition: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM 2-mercaptoethanol, 0.04 mM nucleosidase (dATP, dGTP, dCTP), 0,03 mM Biotin-16-dUTP, 10U DNA polymerase 1 from E. coli, 5-10 μg DNA AZ and 2 μg DNA of recombinant plasmids. The volume of the reaction mixture of 50 µl. The reaction is performed for 1 hour at a temperature of 15°C. the Reaction is stopped by addition of EDTA (50 mM), labeled DNA recombinant plasmids are precipitated with ethanol, dried and dissolved in hybridization mixture (55% formamide, 2×SSC). Store in the dark at -20°C.

Hybridization of fluorescent labeled nucleotides recombinant DNA alpha R1-13 with metaphase chromosomes in situ carried out by the following procedure: labeled recombinant plasmid at a concentration of 10-20 ng in 10 μl of hybridization mixture consisting of 55% solution of formamide in 2×SSC, placed on cytological preparations containing fixed metaphase chromosomes or interphase nuclei, cover with a cover glass 22×22 mm and is heated at a temperature of 75°C for 5 min for simultaneous denaturation of DNA and recombinant DNA; followed by hybridization at 42°C for 4-18-'clock in the wet chamber. Drugs after conducting hybridization washing the Ute in hybridization mixture (50% formamide, 2×SSC) at 42°C for 3 min and a solution of 2x SSC at a temperature of 42°C for 5 min and then rinsed with buffer solution: 0.1 M NaH2PO4and 0.1 M Na2HPO4(pH 8.0)containing 0.1% tween-20 at room temperature (or at 37°C) for 1 min. Then preparations incubated with Avidya labeled with fluorescein (fluorescein isothiocyanate solution for contrasting coloring), for 30 min at 37°C in a humid chamber, and one product are 20 µl avidin (concentration of 5 μg/ml) and covered with a plate or plastic film "Parafilm". Washed drugs three times for 5-15 min buffer 0.1 M NaH2PO4and 0.1 M Na2HPO4(pH 8.0)containing 0.1% tween-20 at 37°C.

For coloring drugs they put sunscreen solution: 2% 1,4-diazobicyclo-(2,2,2)-octane (DABCO) in 50% glycerol, 20 mm Tris-HCl, pH 8.0 (15-20 μl), 400 ng/ml of the fluorescent dye DAPI (4',6-diamino-2-phenylindol-dihydrochloride), propidium iodide at a concentration of 400 ng/ml and covered with a cover glass (22×22 mm) for microscopic observation.

If microscopy using a conventional fluorescence microscope equipped with the appropriate set of filters. For example, for the simultaneous analysis of hybridization signals (green - FITC) and chromosomes (red - propidium iodide) - Phi is Tr I3, Leitz. For chromosomes and interphase nuclei without hybridization signal filter A, Leitz (DAPI - blue) or filter N 2.1, Leitz (propidium iodide - red color).

If you need to strengthen hybridization signals (using cytological preparations of poor quality - poor fixation, etc.) carry out additional processing of the hybridization signals and immunological amplification.

The medicines are washed in buffer 0.1 M NaH2PO4, 0.1 M Na2HPO4(pH 8.0)containing 0.1% tween-20 for 5-15 min; incubated with biotinylating antibodies to avidin (20 µl blocking solution at a concentration of 5 μg/ml) for 30 min at room temperature, or 20 min at 37°C in a humid chamber, a cover plate made of plastic or film "Parafilm". Further preparations are washed in buffer 0.1 M NaH2PO4, 0.1 M Na2HPO4(pH 8.0)containing 0.1% tween-20 three times for 5-15 min at room temperature or at temperature 37°-45°C and then incubated with Avidya labeled with fluorescein (20 µl blocking solution at a concentration of 5 μg/ml)for 30 min at room temperature, or 20 min at 37°C. Then the drugs again washed in buffer 0.1 M NaH2PO4, 0.1 M Na2HPO4(pH 8.0)containing 0.1% tween-20 three times for 5-15 min at room temperature or at 37°-45°C. is prepared for microscopy. The process of amplification of a weak hybridization signal, if necessary, can be performed 2-3 times.

In situ hybridization on metaphase chromosomes of man shows that the cloned fragment alpha R1-13 localized in pericentromeric regions of the 7th pair of homologous chromosomes and is, thus, a specific molecular marker for a given pair of chromosomes.

Methods of using the obtained recombinant plasmid R1 alpha-13 for identification of chromosome 7 person in metaphase and interphase cells with the purpose of molecular cytogenetic diagnostics are explained in the following examples.

Example 1. Whole heparinized blood of healthy donor plant of 0.015 M NaCl in a volume ratio of 1:3. For the deposition of erythrocytes diluted blood is mixed with 6%solution of dextransucrase-500 in the ratio of 5:1; the mixture defend 30 min at room temperature; all subsequent procedures are performed at a temperature of 0-4°C. the Leukocytes from the supernatant precipitated by centrifugation at 450g for 20 min and washed three times by centrifugation in 0.15 M NaCl (450g, 10 min). The washed cells resuspended in the STM buffer containing 0.5 M sucrose, 50 mm Tris HCl (pH 8.0), 5 mm MgCl2, 0.02 mm EDTA with Triton X-100 at a final concentration of 0.05%. The fraction of nuclei washed three times by centrifugation in gabutero without Triton X-100 (450g, 10 min).

Kernel resuspended in THE buffer (50 mm Tris HCl, pH 8.0; 5 mm EDTA) at the rate of: 100 ml buffer per 1 ml of nuclear sludge, and are lysed by adding sarkosyl to a concentration of 1%. The lysate is incubated at a temperature of 65°C for at least 1 hour to full enlightenment and centrifuged for 60 min at 2000 rpm to remove mechanical impurities. The supernatant is transferred into the cylinder from below by an ultra-filter HM-300 ("Amicon"). The supernatant layer is equal to the volume of THE buffer+1%sarcosyl. The fluid in the cylinder is separated from the anode chamber of the dialysis membrane. The bottom edge of the cylinder with the filter immersed in the cathode chamber; both electrode chambers are filled with buffer containing 89 mm Tris HCl, 89 mm NVO and 5 mm EDTA (pH 8.3). Electrofiltration solution is carried out at 5 mm/cm2within 6-8 hours.

After electrophoretic deposition of DNA on the filter, the liquid is removed from the cylinder, and a jelly-like layer of DNA dissolved in the appropriate volume of 0.1 M.

For preparative selection of plasmid DNA with insert human DNA E.coli cells carrying plasmid, grown in 100 ml LB medium (10 g/l peptone, 5 g/l yeast extract, 10 g/l NaCl) with additives necessary antibiotics (100 μg/ml ampicillin) at 37°C on a rocking chair for 12 hours. Cells precipitated by centrifugation (5000 rpm, 10 min) and resuspended in 10 ml of 50 mm glucose, 50 Tris-HCl (R) - Rev. 8,0), 50 mm EDTA and 5% solution of Triton X-100. To the suspension is added a freshly prepared solution of lysozyme to 5 mg/ml and left at room temperature for 10-15 minutes and Then the volume of the sample was adjusted to 50 ml with buffer without lysozyme and placed overnight in a thermostat at a temperature of 65°C. the DNA-membrane complex and denaturated proteins cells precipitated by centrifugation at 12,000 rpm for 10 min. the supernatant add 100 ág/ml RNA basics and after 2 hours incubation at 65°C hold phenol and then chloroform the deproteinization. DNA from solution is precipitated by an equal volume of isopropyl alcohol (temperature 18°C, 20 min), the precipitate was dissolved in TE. The DNA solution cialiswhat on Sephadex G-50 vs. 0.1×SSC (1×SSC g/l of sodium chloride and 4.8 g/l sodium citrate). This solution is heated at a temperature of 81°C for 10 min and rapidly cooled, add a volume of 1M KCl + 20 mm Tris-HCl pH of 7.1 at 0°C. the resulting mixture was passed through a column with nitrocellulose Nitro-Cell-S (Serva) at the rate of 1 mg DNA per 10 cm3volume of the column. The fractions containing the circular forms of plasmid DNA are pooled DNA of them precipitated by an equal volume of isopropyl alcohol; the precipitate is washed in solutions of 50° or isopropyl 70° ethyl alcohol, dried under vacuum and dissolved in THE buffer to the desired concentration.

DNA recombinant plasmids for srinivasacharyulu clones isolated from 5 ml of overnight culture, precipitating by centrifugation at 5000 rpm for 10 minutes, the precipitate resuspended in 1 ml of 25 mm Tris-HCl (pH 8.0); 20 mm EDTA, 50 mm glucose and 2 mg/ml lysozyme, followed by incubation for 30 minutes at a temperature of 0°C. To cells add 2 ml of 0.2 N NaOH 1% SDS for cell lysis, and after thorough mixing, continue incubation for 5 minutes To the lysate add 1.5 ml of 3 M sodium acetate, gently mix the solution and leave it for an hour at 0°C. The formed precipitate is removed by centrifugation (1200 rpm), nucleic acids from the supernatant precipitated with 2.5 volumes of ethanol (-18°C, 30 min). The precipitate is dissolved in 1 ml of 50 mm Tris-HCl (pH 8.0) + 0.1 M sodium acetate and periostat ethanol. Procedure resultant deposition rates was repeated twice, the final precipitate after drying is dissolved in 100 ál TE.

Endonuclease processing of human DNA and bacterial plasmids is carried out in the buffer REB containing 10 mm Tris-HCl (pH 7.4), 10 mm MgCl2, 10 mm NaCl and 1 mm dithiothreitol (DTT). Full endonuclease hydrolysis of DNA is obtained when the relative concentration of restrictase EcoR 1 5-8 u/µg DNA after the reaction for 2 hours at 37°C. the Reaction is stopped by heating the samples at 70°C for 10 minutes

To obtain genomic clontech human DNA as a vector using bacterial the bacterial plasmid pBR325, carrying the gene for resistance to ampicillin. The presence of a resistance gene for ampicillin plasmid allows on the environment with this antibiotic to select transformed cells carrying the plasmid with the human DNA.

Competent cultures of bacteria for transformation was prepared as follows: 25 ml of culture medium LB inoculant 1 ml of fresh overnight culture of cells of E. coli HB 101 and cultivated on a shaker at 37°C until the turbidity of A=0.4 to 0.5. Cell division is stopped by placing the culture on ice for 10-15 min, followed by centrifugation at 5000 rpm for 10 min in the cold. The precipitate is washed with an equal volume of chilled to a temperature of 0°C solution of 100 mm CaCl2the cells are precipitated and resuspended in 12 ml of 100 mm CaCl2. Cell suspension stand in the cold for 20-30 min, centrifuged, and the sediment resuspended 2.5 ml of 0.1 M solution of CaCl2with 15%glycerol. After incubation, the resulting suspension at a temperature of 0°C for at least 30 min culture can be transformed. It is stored in small portions at a temperature of -80°C, while its competence lasts for 4-5 months. Further, when using 200 µl of competent bacteria cultures at a temperature of 0°C, add 0.1-0.2 ág legirovannoi plasmid DNA and incubated at this temperature for 45-60 minutes Then mix Perrin is placed in a water bath (42°C) for 1-1,5 min and placed on ice. It transformed the culture add nutrient broth LB and pokasivaut her on the rocking chair 2-3 hours at 37°C. Transformants plated on solid medium with 1.5% agar (0.1 ml culture in a Petri dish) with the addition of ampicillin, causing the selection of transformed clones.

30 μl of a solution containing 0.1 to 0.2 μg of plasmid DNA or 0.1 μg isolated from preparative gel fragment, and 1 μl of dnaase-1 of 0.015 M NaCl, 5 mm SAS2, incubated 10 min at room temperature, DNA-ABC-1 inactivate at a temperature of 70°C for 10 min, then added to the mixture of buffer solution, 0.4 M Tris-HCl (pH 7.4); 50 mm MgCl2, 25 mm DTT (up to 1/5 of the final volume), 1 nmol of each of the unlabeled precursors of DNA, 20-40 µci one of the32P - deoxynucleosides with specific activity ON TV/mmol (3000 CI/mmol) (Amersham) and 1 u DNA-polymerase-1. The volume of the reaction mixture, typically, 100 μl of reaction are for 1 hour at 37°C. the Radioactivity of the acid-insoluble fraction of the DNA is measured on the meter LRB 1215 in a special program. The average specific activity of the labeled drug is 2-5×108pulse/min/µg DNA.

To identify inserts of human DNA in the hybrid clones of bacterial colonies, pre-tested for phenotype as recombinant, replica put on nitracel wlasnie filters (Millipore, HAWP)directly on the surface of a solid nutrient medium in Petri dishes and grown them for days at 37°C. the Grown colonies with the filter is transferred onto the surface of a solution of 0.5 M NaOH and leave for 5-10 minutes Podsused filter with the lower side of the filter paper twice for 10 min, treated with a solution of 1 M Tris-HCl (pH 7.5), and then 1.5 M NaCl 1 M Tris HCl (pH 7.5). The air-dried filter was placed in a solution of 2×SSC with proteinase K at a concentration of 50 μg/ml and incubated it for 30 min at 37°C. Then the dried filter is washed in 0.3 M NaCl, and then dried under vacuum at a temperature of 80°C for 1-2 hours.

Before hybridization, the filter is soaked at a temperature of 68°C in a solution of 2×SSC, 0,5% SDS, 0.1% ficol-400 and 0.1% polyvinylpyrrolidone - 350 for 60 to 90 minutes. Following this, the filter dry promacot filter paper and placed in 4-5 ml of hybridization mixture containing 6×SSC; 0.1% of SDS; 10% of dextransucrase-500, 50 μg/ml poly (a) and denatured sample32P-labeled DNA with a total activity of 1-5×106pulse/min Hybridization is carried out at a temperature of 68°C for 18-24 hours. The filters are washed in several changes of solution of 0.5×SSC with 0.5% SDS at a temperature of 68°C for several hours. The washed filters finally dried and placed in a cassette with x-ray film RM-1. After 2-24 hours e is pozicii show radioautographic and the presence of positive signals (sections blackening rounded) identify hybrid clones.

As described put the experience on three filters-replicas with the multicopy EcoRl fragments of human DNA. As a probe for hybridization with the colonies on the filters a sample of altenau human DNA, isolated from preparative agarose gel after electrophoretic separation of the hydrolysate human genomic DNA by endonuclease EcoRl and identification stripe size 680 base pairs. In the specified DNA sample is injected isotope label previously described method of substitution. After hybridization in the colonies, a positive signal is detected particularly clear in the several colonies, one of which was named alpha R1-13.

Preparations of chromosomes prepared from cultures of peripheral blood lymphocytes stimulated to divide with the help of phytohemagglutinin (firm Difco, USA). Cells cultivated in penicillin vials or syringes at a temperature of 37°C in the environment of the Needle with the addition of up to 20% bovine serum within 74 hours. An hour before the end of the cultivation injected colchicine at a concentration of 0.5 μg/ml Cells in the environment of the cultivation transferred into centrifuge tubes and centrifuged for 5 min at 1000 rpm Sediment resuspended in hypotonic solution of 0.07 M KCl and incubated for 10 min at 37°C. Fixation spend methanol-acetic acid fixative (3:1) three times for 20 minutes Prep the rata of chromosomes stored at a temperature of 37°C and used in the experiments no later than 2-3 weeks after preparation.

DNA samples for hybridization on chromosomes in situ mark as isotope-labeled and fluorescent labels, method of substitution. In the first case (isotope method), 100 μl of deionized water dissolve consistently 15 ál of ten buffer solution (0.8 M Tris-HCl, pH 7.4; 0.1 M MgCl2); 0,05 M dithiothreitol, 5 ál of non-radioactive deoxynucleotides in equimolar mixture (0.1 mm of each except for the N3-timeinterest), 5 μl of dnaase-1 (concentration 1 μg/ml), 10 μl of N3-timeinterest (specific activity 114 CI/mol, concentration of 5 MCI/ml), 5 μl of DNA polymerase 1 (concentration of 2 u/μl) and 5 μl DNA (1 μg) sample R1 alpha-13. The volume of the reaction mixture is 150 μl. Reactions are within 20-40 minutes at a temperature of 20°C. the Average specific radioactivity of labeled DNA preparations is about 25×106counts per minute per 1 ág DNA.

Hybridization of labeled radioactive precursor recombinant DNA alpha R1-13 with metaphase chromosomes in situ is carried out with preliminary denaturation for 30 sec at 0,N NaOH, followed by washing for 10 min in solutions of 70° and 96.6° ethyl alcohol. Drugs radioactive DNA samples are denatured in hybridization mixture containing 55% solution of formamide, 10% of dextransucrase-500 and a standard saline solution (2×SSC)for 10 mi is at a temperature of 100°C. Hybridization is carried out at a temperature of 37°C for 17-18 hours. Drugs after conducting hybridization washed in two changes of 2×SSC at 37°C, in two changes of 2×SSC at room temperature in solutions of 70° and 96.6° ethanol for 15 min in each shift. After drying in air the drugs covered by the emulsion type M and exhibit in the dark for 10 days. After developing, the standard apidology developer for 2 min medications washed thoroughly with running water and paint 3%dye solution Romanovsky-Institute in 0.02 M phosphate buffer (pH 6.8) for 10-15 min Radioautography analyzed and photographed under a microscope at magnification ×1000, ×1125.

In the second case, when the tagging of recombinant DNA samples of non-radioactive fluorescent nucleotides, for example Biotin-16-dUTP (Biotin-16-deoxy-uridinediphosphate)prepare the reaction mixture of the following composition: 50mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM 2-mercaptoethanol, 0.04 mM nucleosidase (dATP, dGTP, dCTP), 0,03 mM Biotin-16-dUTP, 10 units of DNA polymerase 1 from E. coli, 10 ng dnaase I and 2 μg DNA of recombinant plasmids. The volume of the reaction mixture of 50 µl. The reaction is performed for 1 hour at a temperature of 15°C. the Reaction is stopped by addition of EDTA (50 mM), labeled DNA recombinant plasmids are precipitated with ethanol, dried and dissolved in hybridiza the ionic mixture (55% formamide, 2×SSC). Store in the dark at -20°C.

Hybridization of fluorescent labeled nucleotides recombinant DNA alpha R1-13 with metaphase chromosomes in situ carried out by the following procedure: labeled recombinant plasmid at a concentration of 10-20 ng in 10 μl of hybridization mixture consisting of 55% solution of formamide in 2×SSC, placed on cytological preparations containing fixed metaphase chromosomes or interphase nuclei, cover with a cover glass 22×22 mm and is heated at a temperature of 75°C for 5 min for simultaneous denaturation of DNA and recombinant DNA; followed by hybridization at 42°C for 4-18-'clock in the wet chamber. Drugs after conducting hybridization washed in hybridization mixture (50% formamide, 2×SSC) at 42°C for 3 min and a solution of 2×SSC at a temperature of 42°C for 5 min and then rinsed with buffer solution: 0.1 M NaH2PO4+ 0.1 M Na2HPO4(pH 8.0)containing 0.1% tween-20 at room temperature (or at 37°C) for 1 min. Then preparations incubated with Avidya labeled with fluorescein (fluorescein isothiocyanate solution for contrasting coloring), for 30 min at 37°C in a humid chamber, and one product are 20 µl avidin (concentration of 5 μg/ml) and served plastinates plastic or film "Parafilm". Washed drugs three times for 5-15 min buffer 0.1 M NaH2PO4+ 0.1 M Na2HPO4(pH 8.0)containing 0.1% tween-20 at 37°C.

For coloring drugs they put sunscreen solution: 2% DABCO in 50% glycerol, 20 mm Tris-HCl, pH 8.0 (15-20 μl), 400 ng/ml fluorescent dye DAPI, propidium iodide at a concentration of 400 ng/ml and covered with a cover glass (22×22 mm) for microscopic observation.

In situ hybridization, radioactive (isotope)and radiative (fluorescence) on metaphase chromosomes of man, shows that the cloned fragment alpha R1-13 is localized only in the pericentromeric region of the 7th pair of homologous chromosomes and is, thus, a molecular marker of the given pairs of chromosomes, specifically marking allocentrism areas.

Example 2. Especially important is the application of the proposed molecular marker 7 pairs of chromosomes in cases when these chromosomes (one of the homologues) affected by the restructuring, that is, the loss or addition of any parcel of chromosomal material, which affects the size of the 7th chromosome and its form.

All procedures for the selection of sample DNA alpha R1-13 and its treatment with isotope labeled for hybridization on metaphase chromosomes in situ, as well as all procedures for the preparation of drugs metaphase chromo is om and conducting hybridization on chromosomes repeated analogously to example 1. The only difference is that in example 1 used drugs normal chromosome set from a healthy donor, male, whereas in example 2, used drugs chromosomal male child (the proband) with the following clinical manifestations: mental retardation, ectrodactylia, brachydactyly, short neck, charactername facial anomalies, transverse furrow on the palm, Gothic sky and irregular teeth. The cytogenetic analysis of cells of the proband was found karyotype with supposedly balanced rearrangement (barycentrically inversion of chromosome 7), i.e. without visible loss of chromosomal material in allocentrism area. It should correspond to the normal phenotype of the child. A similar inversion was detected and his phenotypically normal mother. Traditional cytogenetic methods using differential staining of chromosomes in length proved ineffective. However, after conducting hybridization on metaphase chromosomes in situ using molecular marker alpha R1-13 for 7 of chromosome accurately determine changes in the karyotypes of the proband and his mother was not difficult. In both cases, one of the points gap was localized to the pericentromeric heterochromatin (altinay chromosome 7) and Le is to be recognized directly under the microscope. However, there are some differences between carriers of the inversion in the family. The mother inversion formed two separate block altenau DNA, while the proband with phenotypic abnormalities Altena DNA was hybridities homogeneous with inverted plot. It is known that the heterochromatin formed altenau DNA, represents a transcriptionally inactive structure of the chromosome. Therefore, incorporation altinova block in the sequence generalising areas of the chromosome can affect the epigenetic regulation of gene expression of the corresponding site. Clinical differences between the proband and his mother, most likely can be explained by the effect of the provisions of genes as a result of their unusual move due to inversions and hence, changing the order of raspolojenia genes in the corresponding chromosomal sites. Thus, using molecular marker alpha R1-13 for 7 of chromosome changes in homologous chromosomes affected by restructuring are recognized easily in the analysis under a microscope.

Example 3. An important illustration of the practical application of molecular marker alpha R1-13 for 7 pairs of chromosomes is the experience on the identification of the chromosome in the cell nucleus directly on the picture interphase (non-dividing) nuclei. As you know, the human chromosome is and analyzed only in metaphase of mitosis after special processing, to facilitate the calculation and analysis of the morphology of the chromosomes. The exception is the sex chromosome X, the presence of which in normal women can be set according to the definition in the interphase nucleus compact Taurus chromatin X, formed one of the two X chromosomes in the female set, as well as sex chromosome, which determines how bright fluorescent body, painted quinacrine mustard-gas, in the interphase nucleus of men with normal chromosome set. However, using molecular marker alpha R1-13, specifically marking only the 7th chromosome of man, the availability of these chromosomes in the cell nucleus can be installed without preparation of metaphase chromosomes, only the image analysis of interphase nuclei. This is because the DNA sample alpha R1-13 hybridizes with DNA allocentrism areas 7 pairs of chromosomes and in the case when these chromosomes are despiralization condition at the stage of interphase. In each interphase nucleus can be seen after hybridization of two clusters of tags or signal corresponding to the 7th pair of chromosomes.

All procedures for the selection of sample alpha R1-13 and its treatment with isotopic and fluorescent labeled for hybridization on interphase nuclei, as well as all procedures for the preparation of preparations of metaphase chromosomes of peripheral blood cells and carrying out a hybrid of the organization on the chromosome repeat similar procedures, described in example 1. In each interphase nucleus found two clusters of granules labels (two clusters) with isotope tagging or 2 fluorescent signal when non-radioactive labelling, the corresponding homologous chromosomes 7th pair. In different nuclei of the distance between clusters, and various signals. So, there is a real possibility directly in interphase nuclei to analyze the location of the homologous partners specific pairs of chromosomes in human cells, which is of great scientific interest from the point of view of the functional organization of hereditary structures.

Example 4. Extremely important illustration of the practical use of molecular marker alpha R1-13 is the experience of the presence of the 7-th pairs of chromosomes in the cells of native preparations of chorionic villi in the prenatal diagnosis of the fetus.

All procedures for the selection of sample DNA alpha R1-13, it is processed with isotopic and fluorescent labeled for hybridization on metaphase chromosomes in situ and conducting hybridization on chromosomes repeated analogously to example 1. The difference is that in example 1 are used preparations of metaphase chromosomes prepared from cultures of peripheral blood lymphocytes of a healthy donor male, whereas in example 4 are used in native medicines met the phase of the chromosomes of the cells of the chorionic villi.

Native preparations from cells of the chorionic villi obtained as follows: samples of chorionic villi washed in three changes of isotonic (0.9% sodium chloride solution) and put it on for 5-10 min in a 60% solution of acetic acid at room temperature, then put ("print") samples of chorionic villi subject to clean the glass. Fixation spend methanol-acetic acid fixative (3:1) three times for 20 minutes chromosome is placed in a thermostat (37°C) and used in experiments within 1-2 hours after cooking.

The cytogenetic analysis of preparations of cells of the chorionic villi in the fetus was discovered male karyotype with a ring chromosome, presumably ring chromosome 7 (r7). It should be noted that cytogenetic analysis of metaphase chromosomes in native preparations of cells of the chorionic villi is often difficult because of the unsatisfactory state of the metaphase plate. However, this method is used in the practice of prenatal diagnostics, when to get the cytogenetic analysis of the need for 3-4 days in the first trimester of pregnancy. Traditional cytogenetic methods using differential staining of chromosomes in length was in this case ineffective. After conducting hybridization on metaphase chromosomes in situ with m the molecular marker alpha R1-13 to 7-th chromosome, set the origin of the ring chromosome in the karyotype, as these chromosomes can be easily identified directly under the microscope by the presence of two signals. In the analysis of these signals on homologous chromosomes were visible one normal 7-th chromosome and ring the 7th chromosome. As a result of molecular studies in fetal circular chromosome 7 (r7). Thus, using molecular marker alpha R1-13 to 7-th chromosome to establish the presence of chromosome 7 pairs in the karyotype of the fetus when performing prenatal diagnosis is not difficult.

Thus, the developed method of marking the 7th chromosome using the cloned sequence of alpha-satellite DNA alpha R1-13 from human genome is an effective tool for detection of the 7th chromosome as standard preparations of normal human chromosomes after cell culture)and in cases rebuilt chromosome 7 couples at different stages of the cell cycle (metaphase and interphase nuclei), as well as cytological native preparations of cells of the chorionic villi (without cultivation). These capabilities extend the scope of chromosomal analysis in the practice of clinical genetics, genetic counseling and prenatal diagnosis in cases of structural electroic 7th chromosomes of human, marker chromosomes, ring chromosomes, as well as the analysis occurring aneuploidy, especially on 7 chromosomes with various forms of cancer. Possible the effective application of the proposed method in the analysis of hybrid cell lines commonly used for cytogenetic mapping of normal and mutant human genes.

Marking the 7th chromosome of man, which provides for the in situ hybridization of chromosomes in metaphase and interphase cells of the test sample and a DNA probe, presents marker plasmid, in conditions of high hardness, characterized in that the denaturation of chromosomal and plasmid DNA are at the same time in situ at a temperature of 75°C for 5 min in hybridization mixture consisting of 55%aqueous solution of formamide in standard saline solution of 0.3 m sodium chloride and 0,03M sodium citrate (2×SSC), followed by hybridization for 4-18 hours at a temperature of 42°C; washed drugs in standard saline solution (2×SSC with 50% formamide at 42°C for 3 min and a solution of 2×SSC at a temperature of 42°C for 5 min; the detection results of hybridization of the DNA probe with the DNA of interphase or metaphase chromosomes carried out using fluorescent microscopy, and as a marker plasmids used recombinant plasmid R1 alpha-13, containing EcoRl-EcoRl fragment of DNA ve the Torah pBR 325 size 5966 pairs of nucleotides and EcoRl-EcoRl fragment altenau DNA 7-th chromosome is human size 680 base pairs.



 

Same patents:

FIELD: medicine.

SUBSTANCE: reactions are conducted in the same PCR-microtube on walls of which there is a probe immobilised. In comparison with standard, the PCR-tubes used have a feature of construction: an extended internal surface, and as consequence - a great sorption capacity. A process of selective amplificate sorption on the test tube walls occurs after each anneal stage: temperature decreasing leads to hybridisation of amplicon chain not only among themselves, and with probes preliminary immobilised on the test tube walls. Using the similar PCR-microtubes enables increasing sensitivity of an immune-enzyme assay of amplicons and decreasing a number of polymerase chain reaction cycles. The fixed analytical signal will characterise both the presence of the required DNA, and show its concentration in the sample.

EFFECT: offered test system can be adapted for any existing PCR-technique, does not require special instrumentation and is reliable, simple and efficient for all parameters.

6 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: faeces sample is examined in patients with acute enteric infections by bacteriological and microscopical methods. One faeces sample of the patient is concentrated by acetate sedimentation, and the produced sediment is used to prepare simultaneously 3 smears: the first one is stained with Ziehl-Nielsen carbolic fuchsin for acid-fast intestinal protozoa indication, the second one is stained with 1% Lugol's solution for lamblia detection, the third one is stained with polyvalent adsorbed serums in an indirect immunofluorescence reaction for salmonella indication, and is observing specific luminescence, the salmonellosis-protozoal acute enteric infection is diagnosed. 1-4 cells in a field of view enable to diagnose a mild form, and 5-12 cells - a severe infection.

EFFECT: use of the declared method allows producing results already in 1-1,5 h after sampling the material and providing higher diagnostic accuracy ensured by quantitative analysis of adhesive activity of simultaneously examined faeces samples, both for salmonellas, and for intestinal protozoa.

2 ex, 1 dwg

FIELD: medicine.

SUBSTANCE: urine is sampled, centrifuged that is followed by solid-phase extraction with Oasis HLB sorbent with using 100% acetonitrile as an extraction fluid for dimethyl terephthalate extraction. Said solid-phase extraction is conducted by consequent passing 100% acetonitrile, distilled water, the urine sample after centrifugation, distilled water, 20% aqueous acetonitrile and 100% acetonitrile as the extraction fluid through the sorbent; then the prepared extract is analysed by liquid chromatography with using as a mobile phase mixed acetonitrile and water in the at the varying ratio 25:75 vol. % to 90:10 vol. % respectively in a gradient mode which is enabled with combining chromatography by supplying at first the mobile phase containing mixed acetonitrile and water in the ratio 25:75 vol. % for 10 minutes. Then increasing the acetonitrile concentration in the mobile phase to 90 vol. % for 5 minutes and passing such mobile phase for another 5 minutes is followed by decreasing a volume amount of acetonitrile to 25 vol. % for 5 minutes and passing such mobile phase through a column for 10 minutes, while an amount of dimethyl terephthalate is determined by a calibration chart.

EFFECT: high sensitivity of the method combined with selectivity and availability for routine analyses.

5 cl, 6 tbl

FIELD: medicine.

SUBSTANCE: amniotic fluid of a pregnant woman on her 16-19 weeks of pregnancy is analysed for the concentration of arginine and praline by capillary electrophoresis. An aspect ratio is calculated, and if its value is equal to 3.2 and higher, foetus growth inhibition is diagnosed.

EFFECT: higher diagnostic accuracy of foetus growth inhibition.

2 ex

FIELD: medicine.

SUBSTANCE: substance of the method for prediction of a CNS pathology in newborns consists in the fact that that blood of a pregnant woman on her 3rd trimester is examined for a nitrogen oxide. If the blood NO concentration is 6.12 mcmol/l and more, the developing CNS pathology is predicted in a newborn.

EFFECT: use of the declared method enables effective screening assays of the pregnant women for identification of the CNS pathology in newborns.

2 ex

FIELD: medicine.

SUBSTANCE: peripheral blood lactate level is measured. If the postischemic lactate level exceeds 10 mmol/l, reperfusion syndrome is diagnosed.

EFFECT: improved diagnostic accuracy.

5 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: substance of the laboratory diagnostic technique for sepsis consists in a quantitative analysis of blood serum for nonprotein microorganism metabolism products, particularly n-hydroxyphenyllactic (HPLA), phenyllactic (PLA), n-hydroxyphenylacetic (HPAA) and homovanillic (HVA) acids. If the contents of said four acids listed above synchronously excess the a healthy level by 5 times or more, sepsis is diagnosed.

EFFECT: use of said technique allows higher reliability of diagnosing sepsis in the patients with hyperthermia of an uncertain aetiology, in impaired state of the patients in hopital, in the intensive care patients, and in surgical postoperative complications.

4 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention describes a method of quantitative evaluation of blood acetic, propionic, isobutyric, butyric, valeric, isocapronic and capronic acids by gas chromatography analysis wherein a blood sample is acidified with 1 % sulphuric acid to pH 2-3, evaluated acids are extracted with isobutyl alcohol volume of which is related to the blood sample volume as 1:1. The protein separation is enabled by centrifugation. 2-3 drops of 0.4 % alkali is added, and the extract is evaporated dry, further the solid residue is added consistently with 1 % sulphuric acid and isobutyl alcohol that is followed with gas chromatography separation of the mixed acids in a capillary column with a flame ionisation detector, and the amount of each acid is evaluated by a calibration diagram.

EFFECT: higher sensitivity and accuracy of the method of quantitative evaluation of acetic, propionic, isobutyric, butyric, valeric, isocapronic and capronic acids if found in blood together.

5 cl, 1 ex, 4 tbl

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely, to gastroenterology. For non-invasive diagnostics of fibrosis and cirrhosis in case of HBV and HCV infections complex ultrasonic examination of liver and spleen tissue is carried out. Additionally duplex scanning with colour Doppler mapping of porto-hepatic region vessels is performed and quantitative indices of hemodynamics of rate of blood flow in vessels, including splenic vein, are determined. Blood test is analysed and used to determine number of platelets, biochemical blood tests are performed, most significant for determination of disease degree indices of coagulogram are taken. After that, discriminant analysis of obtained characteristics and indices is carried out, and taking into account age and experimentally obtained coefficients, total value of two canonical discriminant functions F1 and F2 for HCV and HBV is calculated. Further, by obtained in empiric way territory map position of point for calculated by patient's concrete indices values F1 and F2 for cases of HCV-infection and HBV-infection is determined. Depending on point location on territory map case of mild fibrosis, severe fibrosis or liver cirrhosis is diagnosed.

EFFECT: method increases reliability of fibrosis and cirrhosis diagnostics in case of HBV and HCV infections.

10 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: peripheral blood thrombocytes of women suffering gestosis of various severity levels on their 32-38 weeks of pregnancy are analysed for the activity of glutathione reductase (GY), NADF-dependent glutamate dehydrogenase (NADFGDG) and NADF-dependent isocitrate dehydrogenase (NADFICDG). A nicotine amide adenine dinucleotide phosphate transfer coefficient (NTC) represented by the relation of the GY activity to a product of the NADFGDG and NADFICDG activities is calculated. At the NTC value is equal to 1.3 and lower, the newborn's Apgar score is predicted to be equal to 6 and less, and the NTC value exceeding 1.3 provides the Apgar score being 7-10 points.

EFFECT: more accurate prediction of the newborn's state.

2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: reactions are conducted in the same PCR-microtube on walls of which there is a probe immobilised. In comparison with standard, the PCR-tubes used have a feature of construction: an extended internal surface, and as consequence - a great sorption capacity. A process of selective amplificate sorption on the test tube walls occurs after each anneal stage: temperature decreasing leads to hybridisation of amplicon chain not only among themselves, and with probes preliminary immobilised on the test tube walls. Using the similar PCR-microtubes enables increasing sensitivity of an immune-enzyme assay of amplicons and decreasing a number of polymerase chain reaction cycles. The fixed analytical signal will characterise both the presence of the required DNA, and show its concentration in the sample.

EFFECT: offered test system can be adapted for any existing PCR-technique, does not require special instrumentation and is reliable, simple and efficient for all parameters.

6 cl, 1 dwg, 1 tbl, 1 ex

New corn plants // 2423528

FIELD: agriculture.

SUBSTANCE: during selection by markers, a set of alleles related to loci of quantity tokens (QTL) that contribute to expression of certain phenes of economic value is introduced into a corn idioplasm. The criteria are selected among grain crop capacity, grain moisture when picked, early and late root fit, stem drowning, frequency of common rust, frequency of ear rotting caused by Fusarium (ear wilt), resistance to Sulcotrione and panicle structure. Invention also relates to the method of such plants production, and also to methods of analysis and screening to identify plants with a required profile of alleles.

EFFECT: improved method of new corn plants production.

33 cl, 1 dwg, 19 tbl

FIELD: medicine.

SUBSTANCE: synthetic oligonucleotides for indentifying DNA of Torque teno virus of all known genotypes are disclosed. Primers are combined in a set for DNA identification in blood and other biomaterials of the infectious agent of the latent viral infection Torque teno virus of Circoviridae family by polymerase chain reaction.

EFFECT: invention allows reliable identification of said virus in a biological material.

FIELD: medicine.

SUBSTANCE: analysed sample is studied simultaneously by two methods: the first one is a indirect immunofluorescence (IIMF) reaction, while the second one involves a polymerase chain reaction (PCR); the IIMF provides using monoclonal antibodies "BCKK" (P-384D and 434D). The antibodies interact with the capsular antigen F1 specific for Y.pestis species, or the plasmid-temperature-independent surface protein PFV which is found in all Y.pestis strains and rare R-form Y.pseudotuberculosis strains. The bacteria luminescence shows the presence of native or fraction-less Y.pestis bacteria, or typical and PFV-atypical Y.pseudotuberculosis strains in the sample. The PCR is conducted by two pairs of primers vlm33for/ISrevl754 - specific for Y.pestis species, and JS - specific for Y. pseudotuberculosis species. The values derived with the first pair of the primers are estimated as positive if observing the amplicons in 400 bps, and with the second pair if observing the amplicons in 223 bps; the analysed sample is identified by the matching the IIMF and PCR values with the reference strains.

EFFECT: method provides quick identification and high-reliability differentiation of all strains.

7 tbl, 6 ex

FIELD: physics.

SUBSTANCE: method for bioindication of water bodies involves collecting samples of planktons inhabiting in a water body, determining the contamination level by analysing said samples and assessing the analysis results. The contamination level is determined via phylogenetic analysis of ribosomal RNA genes (18S rRNA) of planktons in the sample. Phylogenetic trees built from the conservative 18S rRNA gene are determined and evolutionary relationships of the analysed object with other saprobionts are identified. Analysis results are assessed as follows: at high (over 85%) value of bootstrap support of clusters containing the analysed planktons and resistant saprobionts, the following conclusions are made: resistant indicator organisms xeno- or oligosaprobic (or exclusively xenosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in a safe ecological state and there is no threat of negative anthropogenic action, if resistant indicator organisms oligo- and mesosaprobic (or exclusively oligosaprobic) of the water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unstable (transition from safe to unsafe state) ecological state, is under insignificant anthropologic load, is capable of self-recovery and does not need additional environmental protection measures, if resistant indicator organisms meso- and polysaprobic (or exclusively mesosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unsafe state and is under considerable anthropologic load, natural capability of self-recovery is insufficient and the water body needs environmental protection measures, if resistant indicator organisms of polysaprobic water bodies and the analysed plankton merge into one cluster, it is concluded that there is a local ecological disaster and there is need for urgent recovery measures.

EFFECT: high reliability of the biomonitoring result for use without territorial limit, independent of the geographical location of the investigated water body.

3 ex

FIELD: medicine.

SUBSTANCE: bacteria freely immobilised on a slide are processed with a lysis solution containing an ionogenic protein-denaturing detergent and EDTA. Further, DNA-nucleoid is dry heating and microwave incubation stabilised. DNA is stained by high-sensitivity DNA-specific fluorochrome, in conclusion DNA integrity is evaluated. Offered is a kit for evaluating the DNA bacteria integrity which comprises all the components required.

EFFECT: invention can be used for determining the bacterial DNA fragmentation levels by a precise express technique.

16 cl, 11 dwg, 5 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: offered invention can be used for the quantitative determination of alive bacteria. The offered method involves PCR-based product amplification with using the primers able to specific hybridisation with rRNA of a bacterium of interest. It is followed with the analysis of cell number of the alive bacterium of interest taking into account PCR cycle number.

EFFECT: invention allows precise determination of cell number of the alive bacterium of interest in a test sample.

8 cl, 7 dwg, 4 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: it is described that immunogenicity of hemagglutinin (HA) molecule of influenza virus can be increased by substituting amino acids in the HA sequence. Substituting specific residues in HA such as asparagine introduction in position 223 in HA H5 allows ensuring more sensitive hemagglutination inhibition (HI) test provided by changing receptor specificity and/or ability to antibody-antigen linkage. The HA molecules having such substitutions can find application in creating diagnostic prototype viruses.

EFFECT: improved influenza virus vaccines.

9 cl, 3 dwg, 7 tbl, 7 ex

FIELD: agriculture.

SUBSTANCE: specified plant is a plant of Cucumis sativus type and includes at least one area at one chromosome, which gives resistance to closterovirus, and at least one area, which gives resistance to powdery mildew. The area of the chromosome, which gives resistance to closterovirus, is linked to at least one marker selected from the group, made of markers E16/M50-244, E16/M50-188, and E11/M48-251. The area of the chromosome, which gives resistance to powdery mildew, is linked to at least one marker selected from the group made of the following components: a marker of single-nucleotide polymorphism 39TG in SEQ ID NO:1, a marker of single-nucleotide polymorphism 29GA in SEQ ID NO:2, a marker of single-nucleotide polymorphism 193CT in SEQ ID NO:3, mutation of an insert 5'-AATTT-3' in position 221 in SEQ ID NO:4, and markers E16/M50-F-194, E11/M48-F-251, E23/M38-M001, E23/M40-M003, E24/M46-M002, E24/M46-M003, E12/M91-M003, E26/M43-M003, E14/M59-F-134 and E14/M59-F-200.

EFFECT: production of the plant resistant to closterovirus and to powdery mildew of cucumbers.

25 cl, 8 dwg, 4 tbl

FIELD: medicine.

SUBSTANCE: elaborated is method of obtaining factor, which takes part in process of control of appetite and/or body weight. Also described are genes, obtained by said method, polypeptides, coded by said genes, intended for treatment, control or diagnostics of diseases, associated with eating disorders and/or control of body weight. Invention also relates to substances, which inhibit activity of said genes or said polypeptides, intended for treatment, control or diagnostics of diseases, associated with process of appetite and/or body weight control.

EFFECT: using tiasolidindions, possessing PPARγ-agonistic activity, it is possible to obtain genes and polypeptides, involved into regulation of appetite and/or body weight reduction.

27 cl, 41 dwg, 35 ex

FIELD: microbiology.

SUBSTANCE: invention represents plasmid vector for transfer of DNA, which comprises sequence coding various fragments of oncoprotein p185neu, which are able to induce immune response in respect to tumors, which hyperexpress p185neu. Invention is also related to pharmaceutical composition on the basis of vector for prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.

EFFECT: invention makes it possible to increase efficiency of prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.

10 cl, 14 dwg, 2 tbl, 2 ex

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