Diagnostic technique for cattle leukemia virus tolerance
SUBSTANCE: method involves allele-specific Nested-PCR with primers which are matched with nucleotide sequences coding amino acids in positions 70-71 of the amino acid sequence. The allele-specific primers E70f1 - 5'-AGAAGGAGATCCTGGAGGATAG - 3' and R71r1 - 5'-CCTGTCCACCTCGGCCCGCCTATC - 3' are matched with a part of BoLA-DRB3 gene located on chromosome 23 (localisation 23q21). They interact only with the nucleotide sequences coding alleles *11, *23, *28 =*7A causing genetic stability to cattle leukaemia. Then sequencing primer Zond 70/71 5'-GCCCGGCTACACCTGT - 3' is used to identify homo- or heterozygosity of an individual by the given alleles. If observing the primers interacting with alleles *11, *23, *28 =*7A, animals are considered to be leukaemia stable, while the absence of interaction with the same alleles can enable to refer to leukaemia unstable, and to neutral.
EFFECT: invention can be used for mass genetic typing of BoLA-DRB3 leukaemia tolerable animals in livestock and commodity economies for animal selection in a nuclear stock.
2 dwg, 4 tbl, 2 ex
The invention relates to biotechnology and molecular genetics, in particular to methods of gene diagnostics resistance of cattle to leukemia.
There is a method of determining the resistance of cattle to leukemia, which consists in the amplification of exon 2 of the gene BoLA-DRB3 (size PCR product is 284 BP) and subsequent analysis of restriction polymorphism of this region using serial processing endonuclease Rsa I, Hae III, Bst YI (Van Eijk, M.J., .Stewart, J.A.Haynes, and H.A.Lewin. 1992. Extensive polymorphism of the BoLA-DRB3 gene distinguished by PCR-RFLP. Anim. Genet. 23:483-496).
There is a method of determining the resistance of cattle to leukemia, which consists in the amplification of exon 2 of the gene BoLA-DRB3 and subsequent analysis of restriction polymorphism of this region by processing restrictase Rsa I, Hae III, BstYI, Bst2UI (kowaluk, NV and other Selection-genetic method of creating highly productive and resistant to the persistent limfotsitoz cattle. / Kowaluk NV, Shostak, VA, Satsuk V.F., Fomenko A.I // Bulletin of the RAAS. - 2005. No. 6. - P.67-69).
The main advantage of these methods is the ability to identify each specific allele of the previously described detection of new alleles.
The obvious drawback of this analysis is the complexity of the differentiation of genotypes, because the restriction fragments may differ from each other neznacit the flax. In addition, these methods require a lot of time and money on analysis.
There is a method of determining the resistance of cattle to leukemia, taken as a prototype, consisting of allele-specific PCR with primers selected for nucleotide sequences coding for amino acids Glu-Arg at positions 70-71 amino acid sequences and the amino acids Val-Asp-Thr-Tyr at positions 75-78 (J Immunol. 1993 Dec 15; 151(12):6977-85. Polymorphism in BoLA-DRB3 exon 2 correlates with resistance to persistent lymphocytosis caused by bovine leukemia virus. Xu A., van Eijk M.J., Park, C., Lewin H.A.).
This method is faster and cheaper than PCR-RFLP analysis, it also allows you to share all of the results into three groups: stable, sensitive and neutral alleles, rather than selecting each specific allele.
However, this method of analysis does not allow to identify Homo - or heterozygosity individuals on detected alleles.
When creating the present invention, the task was to develop a method of diagnosing the resistance of cattle to leukosis virus, providing a mass screening of the population in order to identify animals-carriers of alleles that provide resistance to leukemia.
The technical result of the invention is achieved by a method of diagnosing the resistance of cattle to leukosis virus, including the allele with ecifica Nested-PCR with primers matched to the positions 70-71 in amino acid sequence, wherein the primers are selected so that, identify animals-media only-resistant leukemia alleles, with subsequent determination of Homo - or heterozygosity of these animals according to the alleles by persecutione.
Total for the prototype and the present invention is the use of Nested-PCR with allele-specific primers.
Unlike the prototype of the invention involves mass screening of the population in order to identify using allele-specific PCR animals carrying only desirable alleles (i.e. alleles determining resistance to leukemia), and the subsequent persecutione found samples to establish a Homo - or heterozygosity of individuals on these alleles. The animals that carry sensitive or neutral alleles, not divided into 2 groups.
The prepared PCR products (amplicons) hybridized with sequanorum primer and diagnose the nucleotide sequence of the gene in real-time and simultaneous determination of 4 amino acids (E, R, Q, K) with the use of technology persecutione and identification of Homo - or heterozygosity for alleles determining resistance or susceptibility to the virus leukemia.
Example 1. Oligonucleotide primers were kindly the us on the basis of gene sequence BoLA-DRB3. They amplified fragments of the gene BoLA-DRB3 cattle in length 284 base pairs (BP), 233 BP and 93 BP (gene Bank M, M, M, M, M, M) in two stages. In the first stage, we used the primers l30 and l31 Protocol (table 1.1), in the second stage - l30, Bola32, E70fl and R71rT(the Protocol in table 1.3);
l30 - 5'-ATCCTCTCTCTGCAGCACATTTCC-3'
Bola31 - 5'-TTAAATTGCGCTCACCTCGCCGCT-3';
Bola32 - 5'-TCGCCGCTGCCACAGT-3';
E70f1 - 5'-AGAAGGAGATCCTGGAGGATAG-3';
R71rl - 5'-CCTGTCCACCTCGGCCCGCCTATC-3';
The primers E70f1 and R71r1 were introduced nucleotide polymorphisms, so that annealing was performed only on the alleles determining resistance to leukemia. The primers do not interact (not annealed) with alleles that cause increased sensitivity to the virus, and neutral alleles (Figure 1).
This allows to identify the presence of any potentially resistant leukemia alleles(*11, *23, *28=*7A) in a single reaction. This allele, not causing resistance to leukemia (sensitive and neutral)do not differentiate.
PCR was performed according to the following protocols in the reaction mixture of the following composition: 1×PCR buffer (16.6 mm (NH4)2SO4, 67.7 mm Tris-HCl, pH=8.8, 0.1% (v/v) Tween 20, 1.5 mm MgCl2), 200 mm dNTP, 10 pmol of each primer, 1 Unit Taq polymerase.
The verification of the effectiveness of passing PCR was diagnosed by the method of gel-electro is oresa, applying 5 µl of amplificate in 3% agarose gel electrophoresis were separated in 1×TAE buffer for 20 min at 100 V and were detected under ultraviolet light (UV).
Next, samples that showed electrophoresis 3 fragments of appropriate length (284, 233 and 93 BP)was subjected to another PCR Protocol (table 1.2), the effectiveness of the passage which was also checked using agarose gel electrophoresis.
The remaining 30 μl of amplification was mixed in a round bottom plastic 96-well-plates with 3 ál of sepharose (Streptavidin Sepharose™, 17-5113-03) and 57 μl of binding buffer (Binding Buffer, 40-0033), incubated at room temperature for 10 min, rapidly mixing on a shaker at 1400 rpm.
Using vacuum workstation (Vacuum Prep Workstation (220-240 V), 60-0207) associated with sepharose matrix PCR products were transferred to the preparation filters and then washed for 10 seconds in 70% ethanol, 0.2 M NaOH (DNA denaturation), 1× wash buffer (Wasching Buffer, 40-0035). After disconnecting vacuum samples 5-10 sec was suirable in the prepared thin-walled plastic 96-well plates (PSQ 96 Plate Low, 40-0010)containing in each well, 45 ál of Annealing Buffer (40-0036) in a mixture of 15 picomoles Sequeira primers selected to mutations in amino acids 70 and 71 of the gene BoLA-DRB3 cattle. As securitysage we have chosen primer, 5'-horse is which Machen Biotin, l30 bio 5'-BIOATCCTCTCTCTGCAGCACATTTCC-3', and developed an analysis scheme that allows simultaneous determination of the polymorphism of the amplified gene BoLA-DRB3 cattle in positions 2 codons 70 and 71.
The thus prepared sample was placed in a thermostat, incubated for 2 min at 90°C for hybridization Sequeira primers to single-stranded DNA-matrix, after cooled at room temperature for 10 minutes
The genodiagnostic conducted on automatic pyrosequencer PSQ™ 96MA SYSTEM (Pyrosequencing AB) using reagents from a set PSQ™ 96 SNP Reagent Kit 5×96 (40-0023) in real-time. Processing raw data persecutione studied SNP was performed using the software PSQ96MA SNP software v.2.0 (Figure 2).
So determined for each animal alleles summed up in a Microsoft Excel spreadsheet. The resulting matrix of genotypes served as a basis for statistical analysis of the results.
Example 2. The developed system was tested on 344 animals black and white breed (Table 3).
The survey revealed that 20,93% of animals are carriers of resistant alleles, while 4,07% are homozygous for these alleles. When diagnosing leukemia virus by PCR among all carriers of resistant alleles were detected only 6,94% of patients, while SRT is t be noted, among homozygotes carriers of the virus have been identified.
Among heterozygotes were identified 5 animals-carriers of the virus, that is to 8.62%.
All homozygotes and heterozygotes for sustainable alleles were REED-negative.
Using the developed system were also analyzed DNA samples from 30 of the bulls of breeds Holstein root (red-spotted, black and white, Ayrshire), published on 6 plempredpriyaty Russia (Table 4).
It was found that 13,33% of the animals are carriers of resistant alleles, while 6,67% were homozygote on these alleles. All animals were free from provirus leukemia.
The information obtained shows the feasibility of using the developed system for the characterization of the gene pool breeding animals.
The invention allows to reduce the costs of working time, labour and money for testing the resistance of cattle to leukemia by reducing the number of the analyzed nucleotide sequences.
The invention can be used for mass genotyping of animals resistant leukemia in BoLA-DRB3, breeding and commercial farms for the selection of animals in the nucleus.
|The PCR protocols|
|Bola 1 table 1.1||Bola 2 psq table 1.2|
|The main solution||Components||The working solution||ál||The main solution||Components||The working solution||ál|
|10×||The buffer||1×||1,5||10×||The buffer||1×||3,5|
|2 mm||dNTP||2||1,5||2 mm||dNTP||2||1,75|
|50 pmol/ál||Boa||10 pmol||0,2||50 pmol/ál||VOA bio||10 pmol||0,2|
|50 pmol/ál||Boa||10 pmol||0,2||50 pmol/ál||Boa||10 pmol||0,2|
|100 mm||MgCl2||0,2||100 mm||MgCl2||0,1|
|5 U/µl||Taq||1U||0,1||5 U/µl||Taq||1U||0,1|
|DNA||1||The product of the 1st PCR||1,5|
|Bola 2 al.sp. Table 1.3|
|The main solution||Components||The working solution||ál|
|50 pmol/ál||Boa||10 pmol||0,2|
|50 pmol/ál||Boa||10 pmol||0,2||50 pmol/ál||E70fl||10 pmol||0,2|
|50 pmol/ál||R71rl||10 pmol||0,2|
|The product of the 1st PCR||1,5|
|Temperature-time mode Rea the Nations amplification|
|The first reaction amplification||The second reaction amplification|
|Temperature||The number of cycles||Time||Temperature||The number of cycles||Time|
|95°C||1||7 min||95°C||1||7 min|
|94°C||10||1 min||94°C||30||1 min|
|60°C||2 min||52°C||30 sec|
|72°C||1 min||72°C||1 min|
|72°C||1||7 min||72°C||1||7 min|
|The distribution of animals of black-motley breed depending on the genotype of the gene BoLA-DRB3|
|The genotype for the gene BoLA-DRB3||The frequency of occurrence of genotype ER||Diagnosis provirus PCR||The results of REID|
|Resistant alleles (ER), including :||72||20,93||5||6,94||67||93,06||0||0|
|Other alleles||272||79,07||56||20,59||216||79,41||10||of 3.56|
|Just herd||344||100,00||61||17,73||283||82,27||10||of 3.56||Table 4|
|The genotypes of bulls gene BoLA-DRB3 contained in 6 plempredpriyaty Russia|
|The genotype for the gene BoLA-DRB3||The frequency of occurrence of genotype ER||Diagnosis provirus PCR|
|Resistant alleles (ER), including :||4||13,33||0||0,00||4||100,00|
Method for diagnosis of resistance of cattle to leukosis virus, including allele-specific Nested-PCR with primers selected for nucleotide sequences coding for amino acids in which Aziziyah 70-71 amino acid sequence,
wherein the allele-specific primers 70fl - 5'-AGAAGGAGATCCTGGAGGATAG-3' and R71r1 - 5'-CCTGTCCACCTCGGCCCGCCTATC-3', select the part of the gene BoLA-DRB3, located on 23 chromosome (localization 23q21):
they interact only with nucleotide sequences encoding alleles*11, *23, *28=*7A, causing genetic resistance to leukemia in cattle and forth using securitysage primer Zond 70/71 5'-GCCCGGCTACACCTGT-3' define Homo - or heterozygosity individuals on these alleles, while the interaction of primers with alleles*11, *23, *28=*7A animals referred to as resistant leukemia, and in the absence of interaction with the same alleles, they can be defined as unstable to leukemia, so and to neutral.
SUBSTANCE: set contains species-specific oligonucleotide primer pairs and appropriate fluorescent-marked probes for conducting one-stage instant identification of several human-pathogenic Orthopoxviruses (VARV, MPXV, CPXV and VACV) by means of real-time multiplex PCR.
EFFECT: invention is intended for instant diagnostics of human and animal Orthopoxvirus infections by real-time multiplex PCR.
10 dwg, 2 tbl, 4 ex
SUBSTANCE: tissue is homogenised in a buffer and centrifuged at 105000 g for 60-90 min at 0-4°C to produce a cytoplasmic fraction which is then incubated with 10 mM of phosphocreatine and 10 mcg/ml of phosphocreatine kinase for 25-45 minutes at 35°C. Cytoplasmic fraction proteins are divided by ammonium sulphate at three stages, at the first stage ammonium sulphate is added to 38% of saturation and centrifuged to isolate a precipitate containing a 26S-proteasome pool, at the second stage, a supernatant is added with ammonium sulphate to 42 % of saturation and centrifuged to isolate a precipitate containing ballast proteins, at the third stage to the supernatant is added with ammonium sulphate to 70 % of saturation and centrifuged to isolate a precipitate containing a 20S-npoteasome pool. Ammonium sulphate is added in portions during 20 min on a magnetic stirrer and further mixed for 20 minutes.
EFFECT: invention allows dividing native 26S- and 20S-proteasomes and isolating them in those amounts they exist in living cells, with preserving at most an undamaged 26S-proteasome structure.
3 cl, 1 ex
SUBSTANCE: there are offered versions of antibodies and their antigen-binding IL-13, particularly human IL-13 specific fragments. There are described: a pharmaceutical composition, a pharmaceutical compound of the antibody, versions of coding and hybridising nucleic acids and expression vectors. There are offered versions of: cells and methods of producing the antibody, methods of treating IL-13 associated disorders. A method of IL-13 detection in a sample is described.
EFFECT: use of the invention provides new IL-13 antibodies with KD about 10-10 M which can be used for diagnosing, preventing or treating one or more IL-13 associated diseases.
87 cl, 37 dwg, 5 tbl, 6 ex
SUBSTANCE: method includes analysing aliquots of said sample by one or more methods of protein description specified in the chromatography. The method is based on genetic analysis techniques specified in RFLP and T-RFLP. These methods can be applied both separately, and in a combination. The offered methods allow obtaining the information on the presence and fractions of various individual proteins or coding sequences. The obtained information can be used for evaluating stability of a polyclonal cell line in process, and also estimating a structure of various parties of end polyclonal products.
EFFECT: methods allow describing the composition consisting more than of 10, 20 or greater number of antibodies.
16 cl, 18 dwg, 18 tbl, 14 ex
SUBSTANCE: composition includes at least three oligonucleotide probes and enables simultaneously determining a level of PSMB4, FCER2 and POU2F2 genes expression. The oligonucleotide composition under the invention is presented to be used, including as a part of a microchip, in a method for prediction of a developing disease in a subject suffering chronic lymphatic leukemia that involves analysing a level of expression of at least three named genes in patient's blood samples.
EFFECT: higher efficacy of the composition.
8 cl, 5 dwg, 16 tbl, 5 ex
SUBSTANCE: method provides recovery of DNA of an analysed strain followed by PCR with using nucleotide primers of terC, ilvN and inv genes of the following sequences: 89-S - AATCAAATCTCGCCCAGC, 89-As -GCTGCGTATCATTTCACC; 45-S - AGTGGTCTGCTTCTCTGG, 45-As -CGGCATACACAGAATACC; inv839 - TACCTGCACTCCCACAAC, inv1007 -CCCATACGCTGATCTACC. The analysed strains are differentiated by matching the sizes of the derived fragments of terC, ilvN and inv genes with the similar fragments in typical strains of principal and nonprincipal plague agents.
EFFECT: invention allows quick, effective and reliable differentiation of the strains.
1 tbl, 3 ex
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.
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
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
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, psychiatry.
SUBSTANCE: one should isolate DNA out of lymphocytes of peripheral venous blood, then due to the method of polymerase chain reaction of DNA synthesis one should amplify the fragments of hSERT locus of serotonin carrier gene and at detecting genotype 12/10 one should predict the risk for the development of hallucino-delirious forms of psychoses of cerebro-atherosclerotic genesis.
EFFECT: more objective prediction of disease development.
FIELD: biotechnology, medicine, proteins.
SUBSTANCE: invention describes new polypeptide in isolated form relating to subfamily of superfamily human immunoglobulins (Ig-Sf). This polypeptide shows at least 70% of homology level with amino acid sequence of murine molecules CRAM-1 or CRAM-2 regulated by the confluence of adhesive (figures 3, 6 are represented in the claim). Also, invention relates to antibodies showing specificity with respect to the polypeptide. Antibodies and soluble polypeptide can be used for treatment of inflammation and tumors. Invention describes polynucleotide or oligonucleotide encoding the full-size polypeptide or its moiety and represents primer, probe, anti-sense RNA and shows the nucleotide sequence that is identical conceptually with human CRAM-1. Invention provides preparing new adhesive proteins from superfamily Ig-Sf that are regulated at the transcription level in endothelium by effect of tumors. Invention can be used for treatment of different diseases, in particular, inflammatory responses.
EFFECT: valuable medicinal properties of polypeptide.
19 cl, 33 dwg, 1 ex
FIELD: biotechnology, genetics.
SUBSTANCE: invention relates to methods used for detecting low frequency mutations occurrence in gene encoding cytochrome b. Method involves isolation of DNA from known fungi for constructing oligonucleotide probe or primer. Then polymerase chain reaction (PCR) is carried out for assay of binding the nucleotide probe with amplicon generated by this reaction, or the presence of amplicon is detected that is generated as result of PCR using indicated primers. Invention provides rapid and precise detection of mutations conferring resistance of fungus against fungicide.
EFFECT: improved diagnostic methods for detecting mutations.
24 cl, 18 dwg, 14 tbl, 18 ex
FIELD: genetic engineering, molecular biology, pharmacy.
SUBSTANCE: invention relates to methods of genome screening and can be used for identification of pharmacological agent in vegetable extract. Method is realized by treatment of cells with a vegetable extract, isolation of protein or RNA from these cells, identification of isolated protein or RNA wherein their concentration differs from that in untreated cells and detection of compound(s) in indicated vegetable extracts. Then cells are treated with the found compound(s), protein or RNA are isolated from cells treated with this compound(s) and compound(s) are identified that cause the stimulation or inhibition of expression of protein or RNA wherein their concentration differs from that in untreated cells. Invention provides carrying out the characterization of biological properties of vegetable extract and to detect the individual compound(s) that elicit unknown or disclosed biological property of this extract.
EFFECT: improved identifying method.
FIELD: medicine, molecular biology, polypeptides.
SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.
EFFECT: valuable medicinal properties of polypeptide.
21 cl, 92 dwg, 14 tbl, 24 ex
FIELD: medicine, biology, molecular biology.
SUBSTANCE: invention proposes a new method for differential diagnosis of representatives of family Chlamydiaceae. Method involves isolation of DNA of pathogen, amplification using real-time polymerase chain reaction (PCR) and primers CM1 and CM2 exhibiting specificity to 5'-terminal fragment of gene omp1 followed by post-amplification analysis of curves of PCR-products melting in the presence of nonspecific fluorescent dye SYBR Green I for separation of Chlamydia species and electrophoretic separation of PCR-products. Identification of species is carried out on the basis of differences in PCR-products melting point wherein melting point curves of all fragments of omp1 are characterized by the presence of two peaks reflecting two-stage dissociation of DNA chains in sites with different A/T-saturation degree. Proposed method provides carrying out the differentiation of all species of Chlamydia that are pathogenic for humans. Except for, method provides carrying out the differentiation of Chlamydiaceae causing diseases in animals and also method is simple, rapid and can be used for direct diagnosis of clinical material samples. Invention can be used in medicine, veterinary science and virology for differential diagnosis of representatives of family Chlamydia.
EFFECT: improved method for diagnosis.
FIELD: medicine, biology, molecular biology.
SUBSTANCE: invention proposes a new method for differential diagnosis of representatives of family Chlamydiaceae. Method involves isolation of DNA from pathogen, amplification of target using primers CM1 and CM2 showing specificity to 5'-terminal fragment of gene omp1 and electrophoretic separation of polymerase chain reaction (PCR) products. Electrophoresis is carried out in agarose gel with addition of sequence-specific DNA-ligand - bis-benzimide-PEG. The species belonging of PCR-products is determined by comparison of the migration rate of PCR-products in gel with electrophoretic mobility of control. Proposed method provides carrying out the differentiation of all species of family Chlamydiaceae and method is simple and rapid and can be used for direct diagnosis of clinical material samples also. Invention can be used in medicine and virology for differential diagnosis of representatives of family Chlamydiaceae.
EFFECT: improved method for diagnosis.
2 dwg, 2 ex
FIELD: molecular biology, medicine, biochemistry.
SUBSTANCE: invention proposes a method for assay of mononucleotide changes in the known sequences of nucleic acids. Method involves hybridization with PCR-amplified matrix DNA and the following ligation a tandem on its consisting of tetranucleotide that comprises the diagnosed change and two oligonucleotides of the size 8-10 nucleotides being one of that is immobilized on surface of a solid-phase carrier through a 5'-phosphate linker, and the second oligonucleotide is labeled by 3'-end with biotin label. Then tetranucleotide is hybridized with the matrix DNA chain between two oligonucleotides directly that can be ligated with immobilized oligonucleotide through the 5'-end and with non-immobilized oligonucleotide through the 3'-end. The ligation product is detected by its transformation to enzyme label through the complex with high-affinity enzymatic catalyst followed by development of enzyme label in the presence of chromogenic, luminogenic or fluorogenic substrates. Applying a method provides preparing the simple and highly selective agent used for detection of known changes in gene structure.
EFFECT: improved assay method.
8 cl, 3 dwg, 30 ex
FIELD: genetic engineering, biotechnology, biochemistry, agriculture, food industry, medicine.
SUBSTANCE: invention relates to the transformation of plant with nucleic acid encoding enzyme Δ6-desaturase in C. elegans that results to preparing a plant with enhanced content of gamma-linolenic acid and resistance to cold. Desaturase extracted from the plant can be used for preparing a drug used for treatment of disorder in body associated with deficiency of gamma-linolenic acid in it.
EFFECT: valuable biological properties of genes and desaturases.
36 cl, 9 dwg, 2 ex
FIELD: medicine, hematology.
SUBSTANCE: one should isolate DNA out of peripheral blood lymphocytes due to polymerase chain reaction (PCR) technique of DNA synthesis, carry out genotyping for polymorphism of promoter area of TNF-alpha and TNF-beta gene. While detecting genotype LT*22 being characterized by availability of gene TNF-beta mutation in homozygous state, detect persons predisposed to the development of chronic lympholeukosis, and at certain combinations of genotypes TNF*22/LT*22 or TNF*12/LT*11 in patients with chronic lympholeukosis on should predict an aggressive flow of this disease.
EFFECT: higher accuracy of prediction.
3 ex, 3 tbl