Set of synthetic oligonucleotides for detecting nucleotide sequence of coding portion of des gene and identifying mutations associated with desmin-related cardiomyopathies

FIELD: medicine.

SUBSTANCE: invention refers to molecular biology and can be used in diagnosing cardiomyopathies of various origin Presented is a set of synthetic oligonucleotides for identifying mutations of a coding part of desmin (DES) gene associated with cardiomyopathies. The mutations are identified by detecting a full sequence of the coding part of DES gene. The coding part of DES gene is amplified by means of 7 pairs of synthetic oligonucleotides at the same temperature and annealing time, and the prepared amplification products are sequenced by means of one pair of universal primers.

EFFECT: presented invention enables the sensitive and specific detection of DES gene mutations, with reducing the amplification reaction time, the number of manipulations, the time of agent application for the sequencing reaction, and reducing a probability of a reaction error.

3 cl, 1 dwg

 

The present invention relates to the field of medicine, particularly to cardiology and molecular biology, and can be used to identify mutations in the coding parts of desmin gene (DES) associated with cardiomyopathies of different nature, to determine the tactics of conducting patients destinului cardiomyopathy and risk assessment of disease development in identifying family caused by alterations in desmin cardiomyopathy.

Mutations in the coding parts of the gene DES are the cause of approximately 2% of all cardiomyopathies, up to 70% caused by alterations in desmin cardiomyopathy have a family character. Early diagnosis of such conditions will find timely and adequate treatment.

Cardiomyopathy is a chronic disease of the myocardium associated with impaired function of the heart. One of the genes described in connection with the development of dilated cardiomyopathy is of desmin gene (DES). Desmin, a cytoskeletal protein that belongs to the group of intermediate filaments. It is expressed in all three types of muscle tissue - smooth, cardiac and striated skeletal, being the main representative group of intermediate filaments in Mature muscle cells. Phenotypic manifestations of gene mutations DES may be a cardiomyopathy or distal myopathy, but in some cases it's combined phenotype [Goldfarb LG, Vicart Ρ, Goebel EH and Dalakas MC (2004) Desmn myopathy has. Brain, Vol.127, Pages 723-734].

Gene mutation DES can cause the development of dilated cardiomyopathy and contribute to the development of the disease approximately 2% of cases [Taylor MR, Slavov D, Ku L, Di Lenarda A, Sinagra G, Carniel E, Haubold K, Boucek MM, Ferguson D, Graw SL, Zhu X, Cavanaugh J, Sucharov CC, Long CS, Bristow MR, Lavori P, Mestroni L (2007) Prevalence of desmin mutations in dilated cardiomyopathy. Circulation, Vol.115, No. 10, Pages 1244 - 1251; Kostareva A, Gudkova A, Sjoberg G, Kiselev I, Moiseeva O, Karelkina E, Goldfarb L, Schlyakhto E, Sejersen T. (2006) Desmin mutations in a St. Petersburg cohort of cardiomyopathies. Acta Myologica, Vol.25, No. 3, Pages 109-115].

Identification of genetic determinism, timely diagnosis and treatment of asymptomatic patients with cardiomyopathy - severe chronic heart disease with poor prognosis and high mortality, are important to improve the prognosis and survival of patients.

As one of the manifestations of cardiomyopathies caused by mutations in the coding portion of the gene (DES), is the disruption of the conduction system of the heart and the occurrence of atrioventricular blockades, timely detection of gene mutations DES using genetic methods will allow time to determine the tactics of treatment of a patient and to determine the indications for staging pacemaker.

Desmin - cytoskeletal protein with a molecular weight of 53 kDa, belonging to the class of intermediate filaments and which are specific to the myshechom�Oh tissue. Gene desmin (DES) is located on the long arm of the 2nd chromosome (2q35). In 1996, the gene sequence was deciphered, and was later proven link mutations of desmin with distal myopathy and cardiomyopathy. The gene consists of 9 exons which are located on the site 8363 base pairs, and is highly among vertebrates. Desmin is expressed in striated and smooth muscles and in the myocardium.

To date, described more than 40 mutations in the coding parts of the gene DES, most of which are localized in the C-terminal part of the protein, which corresponds to the 5th and 6th exons.

Identification of mutations in the coding parts of the gene DES recommended for use in practical health care in patients with cardiomyopathy to determine the genetic causes of disease. At present, however, in routine practice, medical labs not screening for mutations in the coding parts of the gene DES, since all known methods of determining the sequence of the coding part of the gene DES require time-consuming, involve a large number of manipulations and lead to a large number of errors due to the complexity of setting reactions.

The prior art discloses a method for determining the sequence of the coding part of the gene (DES), described in: Li D, Tapscoft Τ, Gonzalez O, Burch PE, Quiñones MA, ZoghbiWA, Hill R, Bachinski LL, Mann DL, Roberts R (1999) Desmin Mutation Responsible for Idiopathic Dilated Cardiomyopathy. Circulation; Vol.100, No. 5, Pages 461-464. When implementing this method, genomic DNA is isolated from blood samples of patients, using a standard method of DNA extraction. On a dedicated conduct DNA amplification by polymerase chain reaction (PCR) using 7 pairs of synthetic nucleotides, also called primers designed for the 9 exons of desmin gene. Primer sequences are shown below:

exon 1F, CTGATGTCAGGAGGGATACA;

exon 1R, AGAAGGCAGGTGGTGACAG;

exon 2-3F, CTTTATCACCCGCAACTGTC;

exon 2-3R, TATTCCCAGCCAGAGCCTC;

exon 4-5F, AGGCTCTGGCTGGGAATAG;

exon 4-5R, ATGGCCAAGGTCACAAAGTG;

exon 6F, TTTGGGCTGCTAGTGTCCTC;

exon 6R, ATCAGTAATCTCGAGCCTCC;

exon 7F, ATGGTCTCGATCTCCTGACC;

exon 7R, CCCTTTTCTTCCCTAGCTC;

exon 8F, GAAGTAACAAGCCTGTCTTG;

exon 8R, GATCTCTCTTGCCCACTAGC;

exon 9F, CGTCATCCTGCTAGCACATG;

exon 9R, CTGGCAGTCAAGACAACAGG.

Pnrm carried out using 200 ng of genomic DNA, in the presence of 20 mmol/l Tris-HCl (pH of 8.4), 50 mmol/l KCl, 1.5 mmol/l MgCl2, 50 µmol/l of each deoxynucleotide, 0.2 µmol/l direct primer, 0.2 µmol/l reverse primer and 2.5 units of Taq polymerase in a final volume of 50 µl. For each pair of primers choose your PCR conditions - time and temperature of annealing. As a result get a PCR DNA fragments by size 709, 385, 621, 361, 299, 329, and 859 base pairs, respectively. PCR products are purified using standard techniques. Purified PCR products sequani�comfort on a genetic analyzer ABI 310 (Applied Biosystems) using the kit Big Dye Terminator sequencing kit" company Perkin-Elmer and the above primers.

However, this method is very laborious and time-consuming: because of the different temperature and time of annealing, PCR for each pair of primers should be separate. At the stage of introduction of the reagents into microplates for sequencing much time is spent on verification of compliance made primers and amplification products. At this stage allowed the bulk of the errors leading to the need for re-sequencing of the coding part of the gene (DES). These features make it impossible wide application of said method in routine laboratory practice for screening of mutations in the coding parts of the gene (DES).

The prior art is not revealed a prototype of this invention.

The technical result, which directed the invention is to reduce time wasted in the process of amplification, reducing the number of manipulations on stage sequencing, as well as improving the accuracy and reliability of the diagnosis by reducing the probability of errors in the sequencing of the exons of the gene (DES).

Reducing the elapsed time is achieved as a result of the amplification of all exons of the gene DES simultaneously at the same temperature and time, primer annealing due to the use of the proposed set of primers with seq�valinoti Seq ID NO:1-14.

Additionally, reducing the number of manipulations and consequently reduce the elapsed time and reducing the probability of error is achieved by subsequent sequencing of amplification products obtained with the use of a single pair of universal primers by the addition to the sequences Seq ID NO:1 to 14 with 5'-end sequence of one of those pairs of universal primers.

In particular, such as universal primers can be used straight primer pUC/M13(-21) Seq ID NO:15 and a reverse primer M13(-29) Seq ID NO:16.

The present invention provides a primer set for determining the sequence of the exons of the gene DES by which amplification of all exons can be carried out simultaneously at the same temperature and time of annealing of primers and subsequent sequencing of amplification products can be carried out using one pair of universal primers.

The development of specific primers for the amplification

Selection of specific primers for amplification for DNA fragment is carried out by a method well known to specialists in this field. Work on the creation of primers is usually built as follows:

1) using public and commercial databases of nucleotide sequences in �spruce genome choosing unique plot, containing the area of interest of the researcher.

2) On the basis of the selected area of the genome with the help of special software choose the sequence of the oligonucleotides used for the panorama, as a rule, 2 primer. Many of these programs are freely available, for example, Primer3 (http://frodo.wi.mit.edu/), Primer designing tool (NCBI), PerlPrimer (http://perlprimer.sourceforge.net/).

3) Pair of primers additionally check for polymorphisms in the target sequence region complementary to the 3'-terminal portion of the primer.

4) To the direct sequence of primers with 5'-end is attached a sequence of one of the universal primers, and the sequences of the reverse primers with 5'-end attached another sequence of the universal primer is different from that which is attached to direct primers.

4) Primers are synthesized to order in a specialized service center, for example NPK Synthol.

5) With the help of practical experiments demonstrate the suitability of the selected sequences of primers for a particular purpose.

The choice of primers, which are the subject of the present invention, is characterized in that the primers were selected so that the optimum temperature and time of annealing all primers for exons of the gene DES was the same.

Offer great�Mer for amplification of exons of the gene (DES) is characterized by that contains

1) primers following nucleotide composition:

The exon of the gene DESPrimerSequenceThe sequence number
1F1Des1f1GCTGATGTCAGGAGGGATACSeq ID NO:1
1R1Des1r1GGTCATTGATCGTTGGTGCGSeq ID NO:2
1F2Des1f2ACGCCCTCCTCCTACGGCGCSeq ID NO:3
1R2Des1f2GAAGGCAGGAGGTGACAGCGSeq ID NO:4
2-3FDes2-3fCTGCCTCTACCCAGCACCASeq ID NO:5
2-3RDes2-3rATTCCCAGCCAGAGCCTCASeq ID NO:6
4-5FDes4-5fAAGCCCAGTCATGCCCTACAGSeq ID NO:7
4-5R Des4-5rAGTGAGGGTGTGAACTGCAGACAGSeq ID NO:8
6FDes6fGCCTGCCAGCCCAAAGCTTTCTTTGSeq ID NO:9
6RDes6rTGAGGTAATCAGTAATCTCGAGCCTCCSeq ID NO:10
7FDes7fGGCCGATGGGAGGGTTCTTASeq ID NO:11
7RDes7rAGTGCCCATGCTGGACTCCTSeq ID NO:12
8-9FDes8-9fGAAGAAGTAACAAGCCTGTCTSeq ID NO:13
8-9RDes8-9rTGTGATTTGGAGGACTGAGGCSeq ID NO:14

2) additionally, the sequences of the primers Seq ID NO:1, Seq ID NO:3, Seq ID NO:5, Seq ID NO:7, Seq ID NO:9, Seq ID NO:11, Seq ID NO:13, called "direct primers, with 5'-end attached single universal sequence primer, for example a direct primer pUC/M13(-21) Seq ID NO: 15 and sequences of primers Seq ID NO:2, Seq ID NO:4, Seq ID NO:6, Seq ID NO:8, Seq ID NO:10, Seq ID NO:12, Seq ID NO:14, called "reverse primers�", with 5'-end attached to another sequence of the universal primer, such as a reverse primer M13(-29) Seq ID NO: 16.

In addition to the proposed primers in the reaction mixture for amplification comprises the following components:

- a mixture of deoxyribonucleotides four types (dATP, dTTP, dGTP, dCTP);

- the enzyme Taq polymerase;

- reaction buffer: 100 mmol Tris-HCl, pH 8.8 at 25°C, 500 mmol / l KCl, 20 mmol MgCl2.

The amplification of the exons of the gene DES

Coding part of the gene length DES 2268 pairs of nucleotides consists of 9 fragments, or exons. In the case of small exons proposed set of primers allows amplificatoare 2 entire exon located between the fragments of the non-coding portion of a gene, or an intron. In the case of large exons, such as exon 1, the amplification is carried out in two overlapping fragments. Amplification is carried out at the rate of 7 reactions to one sample of genomic DNA according to the following scheme:

Room
reaction
mixture
The number of the exonUsed pair of primers
11Des1f1, Des1r1
21Des1f2, Des1r2
32-3Des2-3f, Des2-3r
44-5Des4-5f, Des4-5r
56Des6f, Des6r
67Des7f, Des7r
78-9Des8-9f, Des8-9r

The amplification reaction is carried out simultaneously for all reaction mixtures with the use of specialized equipment - amplifier Veriti® Thermal Cycler, Applied Biosystems, USA according to the following temperature regime:

30
No.TemperatureTimeThe number of cycles
195°C5 min1
295°C4530
360°C30
472°C
572°C11 min1

Segrenyova exons of the gene DES

After the reaction, the amplification obtained in the first stage of analysis of the amplification products is used for the reaction sequencing. In the reaction sequencing of amplification products obtained as a result of the first stage of analysis, using universal primers, such as direct primer pUC/M13(-21) Seq ID NO: 15 and a reverse primer M13(-29) Seq ID NO:16. The primers are mixed with the reaction buffer before work, and thus eliminate the step of making different primers in the wells of the microplate, in which will be the sequencing reaction, it significantly reduces the risk to make a mistake while entering the components and reduces the time required to make the chemicals more than doubled.

The sequencing reaction is carried out with a set of labeled (BigDye Terminator Cycle Sequencing Kit, version 3.0, Applied Biosystems, USA) and specialized equipment - amplifier Veriti® Thermal Cycler, Applied Biosystems, USA, according to the following temperature regime:

No.TemperatureTime The number of cycles
195°C5 min1
295°C4530
360°C30
472°C30
572°C11 min1

Electrophoretic analysis of the products of sequencing to produce sequencer ABI PRISM 377, Applied Biosystems, USA. The processing of the data produced using the BioEdit program or other similar software.

The invention is illustrated by the following graphic material.

Fig.1 presents a graphical depiction of the sequencing results obtained using the inventive primers for amplification and determination of the nucleotide sequence of exon 2 of the gene (DES). Found IVS3+1G→A mutation of a gene (DES).

The inventive primer set is used as follows:

1) Obtaining biological material from the patient. As samples for analysis m�may be used tissue biopsies, blood, scrapings of buccal epithelium.

2) isolation and purification of DNA from the original samples. Methods for isolating DNA from biological materials are well known in the art and typically include the stage of cell lysis, DNA isolation and purification. Fast and high quality DNA isolation can be performed using commercially available kits, such as "Flexi Gene DNA kit (not the subject of this patent).

3) Amplification of the obtained preparations of genomic DNA with the use of the proposed set of primers specific to exons of the gene DES, 7 reactions under the same conditions, 1 load of the amplifier. Electrophoretic separation and subsequent purification of amplification products.

4) Sequencing of amplification products obtained in section 3 using a pair of commercially available universal primers, 7 reactions. Purification of the reaction products of sequencing and subsequent capillary electrophoresis.

5) analysis of the results of sequencing reactions using specialized software such as BioEdit.

1. A set of synthetic oligonucleotides to detect�tion of the nucleotide sequence of the coding part of the DES gene and detect mutations associated with cardiomyopathies, including primers following nucleotide sequence:

GCTGATGTCAGGAGGGATACSeq ID NO:1
GGTCATTGATCGTTGGTGCGSeq ID NO:2
ACGCCCTCCTCCTACGGCGCSeq ID NO:3
GAAGGCAGGAGGTGACAGCGSeq ID NO:4
CTGCCTCTACCCAGCAGCCASeq ID NO:5
ATTCCCAGCCAGAGCCTCASeq ID NO:6
AAGCCCAGTCATGCCCTACAGSeq ID NO:7
AGTGAGGGTGTGAACTGCAGACAGSeq ID NO:8
GCCTGCCAGCCCAAAGCTTTCTTTGSeq ID NO:9
TGAGGTAATCAGTAATCTCGAGCCTCCSeq ID NO:10
GGCCGATGGGAGGGTTCTTASeq ID NO:11
AGTGCCCATGCTGGACTCCTSeq ID NO:12
GAAGAAGTAACAAGCCTGTCTSeq ID NO:13
TGTGATTTGGAGGACTGAGGCSeq ID NO:14,

not necessarily United with the universal primers.

2. A set of synthetic�fir nucleotide according to claim 1, in which the sequences Seq ID NO:1, Seq ID NO:3, Seq ID NO:5, Seq ID NO:7, Seq ID NO:9, Seq ID N0:11, Seq ID NO:13 with 5'-end attached to a first universal sequence primer, and by the sequences Seq ID NO:2, Seq ID NO:4, Seq ID NO:6, Seq ID NO:8, Seq ID NO:10, Seq ID NO:12, Seq ID NO:14 with 5'-end attached to the second universal sequence primer, wherein the first and second universal primer using different universal primers.

3. A set of synthetic nucleotides according to claim 2, wherein one of the first or second universal primers used straight primer pUC/M13(-21) Seq ID NO:15, and as another universal primer using the reverse primer M13(-29) Seq ID NO:16.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to AXL signalling pathway inhibitors, and can be used in medicine. What is produced is a soluble AXL polypeptide version free from AXL transmembrane domain, which contains at least one amino acid modification in position No. n, wherein n is specified in 32, 72, 87, 92 or 127 or a combination thereof, wherein n+7 is described by numbering SEQ ID NO: 1 that are wild-type AXL sequences, wherein the above modification increases a AXL polypeptide binding affinity to protein 6 specifically inhibiting the growth (GAS6), which is twice as strong as the wild-type AXL polypeptide affinity. The polypeptide can be fused with Fc fragment and used in a method of treating, reducing or preventing tumour dissemination and invasion in a mammalian patient.

EFFECT: invention enables inhibiting the AXL/GAS6 signalling pathways effectively.

8 cl, 15 dwg, 6 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a set of oligonucleotide primers and fluorescently-labelled probe for identification of Burkholderia pseudomallei and differentiation of the actinobacillus mallei by the method of polymerase chain reaction with fluorescence detection.

EFFECT: invention enables in a short time with high sensitivity and specificity to detect the melioidosis agent and differentiate it from the actinobacillus mallei in samples of pure cultures and biological material.

1 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, molecular biology and biotechnology. What is presented is a method for determining the polymorphism of human GP6 gene coding glycoprotein VI by the polymorphous position rs 1613662 based on recording melting curves with fluorescence-labelled allele-specific oligonucleotide tests.

EFFECT: due to the more reliable determination of variable positions and a possibility to detect in one test tube with the use of standard equipment, the method can be effectively used to diagnose the individual's inherited predisposition with the recording the real-time PCR results.

1 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to internalisation of therapeutic molecules into cell, and can be applied in medicine. Obtained is composition for delivering molecules of nucleic acids into cells, containing at least one peptide with at least 92% identity to GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 2); IREIMEKFGKQPVSLPARRLKLRGRKRRQR (SEQ ID NO: 3); or YLKVVRKHHRVIAGQFFGHHHTDSFRMLYD (SEQ ID NO: 4), bound to one or several molecules of nucleic acids.

EFFECT: invention makes it possible to increase efficiency of delivery of molecules of nucleic acids into mammalian cell due to peptide, capable of internalisation into mammalian cell with efficiency, constituting at least 200% of efficiency of internalisation of peptide TAT, which has amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).

8 cl, 16 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, namely to a method for analysing the applicability of RNA extracted from a tissue or a cell (cells) fixed by a fixative for analysing gene expression. The method involves performing electrophoresis with the above RNA. The method implies stating, if the above RNA complies with the following equation: B/A≤1, wherein A represents the mass ratio (%) of RNA falling within the range from 1,000 to 4,000 nucleotides to the total mass of RNA that is determined by electrophoresis, while B represents the mass ratio (%) of RNA falling within the range from more than 4,000 nucleotides to the total mass of RNA that is determined by electrophoresis. If the above RNA extracted from the tissue or cell (cells) complies with the above equation, it is considered to be applicable for analysing gene expression.

EFFECT: presented invention enables the fast and high-effective determination of RNA applicability for analysing gene expression.

7 cl, 3 dwg, 11 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to cell lysis. A method for selective lysis of animal cells and a device for detecting microorganisms are disclosed. A method for selective lysis of animal cells in a water sample with animal cells, containing or possibly containing microorganisms, includes steps of providing a water sample with animal cells, containing or possibly containing microorganisms, adding to said sample a nonionic detergent and a buffer solution to obtain a solution with pH of about 9.5 or higher, incubating said solution for a period sufficient for lysis of animal cells. The device for detecting microorganisms in the sample consists of a lysis chamber for receiving a water sample with animal cells, a vessel with an alkaline buffer solution with pH 9.5 or higher and a nonionic detergent, or a vessel with an alkaline buffer solution with pH of about 9.5 or higher and a vessel with a nonionic detergent, a filter connected to the lysis chamber for filtering the sample after lysis of the animal cells, an indication chamber for analysing presence of DNA of microorganisms.

EFFECT: present inventions enable to process a sample without considerable dilution thereof and without using enzymatic breakdown of DNA or heat treatment, and also enables to process considerably larger sample volumes and determine lower concentrations of pathogenic microorganisms in a sample.

13 cl, 12 dwg, 8 ex

FIELD: biotechnology.

SUBSTANCE: characterised method comprises carrying out of PCR with use of specific primers to genes vc0497, vc0502 and vc0514 from the island of pandemicity VSP-II. The characterised test system comprises the components for isolation of DNA, the components for carrying out PCR, including, in particular, a primer mix VSPIIreg-F - 5'-TGGAAAGAAGAGCGTTACTGC-3', VSPIIreg-R - 5'-CCCTGTTGATGATGTGATTTG-3' to the gene vc0497, VSPIIpilin-F - 5'-CTGTGATTCGGGCTTTATCGG-3', VSPIIpilin-R - 5'-GCGTAAACTGAGCCAATAAGC-3' to the gene vc0502, VSPIIchem-F - 5'-CTTGATGGAGCGGAGAAAAC-3', VSPIIchem-R - 5'-CGATGAATAGCCTGTTGAAC-3' to the gene vc0514, taken in the ratio 1:1:1:1:1:1, respectively.

EFFECT: inventions enable to differentiate quickly and reliably the toxigenic genetically modified strains to genovariants with low and high epidemic potential.

2 cl, 1 dwg, 2 tbl

FIELD: biotechnology.

SUBSTANCE: method comprises isolation of DNA from lymphocytes in peripheral blood by the method of phenol-chloroform extraction, carrying out of PCR, amplification of 18 parts of gene MYO7A, detection in the denaturing acrylamide gel and sequencing. PCR is carried out using specially selected sequences of oligonucleotides flanking regions of 18 exons of the gene MYO7A with possible content of different mutations.

EFFECT: invention enables to simplify the method and to improve the accuracy of determining mutations of the gene MYO7A, to reduce the time of the study.

3 dwg, 1 ex

FIELD: biotechnology.

SUBSTANCE: proposed primers comprise endonuclease cleavage sites, flanking genomic regions encoding the glycoproteins Gn and Gc, and the nucleoprotein N, for obtaining libraries of genes encoding glycoproteins Gn and Gc and N nucleoprotein of Rift Valley fever virus.

EFFECT: invention can be used in creating a bank of nucleotide sequences encoding immunodominant proteins of Rift Valley fever virus Gn, Gc and N, which can be used for creation of diagnostic and vaccine preparations based on recombinant technologies.

3 ex

FIELD: biotechnology.

SUBSTANCE: characterised oligonucleotide primers are complementary to a specific region of the mig-gene of Mycobacterium avium and have the following base composition: 5'-CGT CAA AAG CGA ACT GCA-3' and 5'-TAA TTC GTT GCC CGA CTC-3'. The method of detecting DNA of Mycobacterium avium comprises DNA isolation, DNA amplification using oligonucleotide primers, transfer of amplification product on the gel followed by detection of the analysis results on the transilluminator. In case of positive reaction a fragment is synthesised, corresponding to the size of 157 bps.

EFFECT: inventions can be used in veterinary diagnostic, scientific and practical laboratories for detection of genetic material of Mycobacterium avium in samples.

2 cl, 1 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry. There are presented pharmaceutical compositions having a concatemer molecule and a kit, as well as using for preparing an agent for immune system modulation or for human's or animal's immune system activity modulation, and a composition according to the invention. The given invention can find further application as an immunomodulatory agent in therapy of various diseases.

EFFECT: what is presented is a concatemer molecule of non-coding nucleic acid containing at least four single-strand sites with non-methylated CG motives for human's or animal's immune system activity modulation.

20 cl, 4 dwg

FIELD: biotechnologies.

SUBSTANCE: invention refers to a method of detection of Mycobacterium tuberculosis isolates resistant to pyrazinamide by detection of mutations in pncA gene, associated with evolving resistance to pyrazinamide, by PCR in real time mode using HRM analysis. It involves amplification in a mixture of equal amounts of tested DNA and wild type DNA using primers: Pnc18U: 5'-TACGCTCCGGTGTAGGCAC-3' and Pnc15R: 5'-GAAGCGGCGGACTACCATC-3', formation of heteroduplexes due to simultaneous co-amplification of wild type DNA and test DNA, where the first PCR stage includes concentration equalisation by quantitative PCR using the same pair of primers (Pnc18U/Pnc15R) with calibration curve generation and use of regression equation.

EFFECT: reliable and fast detection of mutations in pncA gene associated with evolving resistance to pyrazinamide, due to high specificity and sensitivity.

1 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to oligopeptides, containing the sequence NLSSAEVVV (SEQ ID NO:6), in which one or two amino acids can be substituted, possessing the inducibility of cytotoxic T-cells, their pharmaceutical compositions and application for the production of anti-cancer vaccines.

EFFECT: obtaining pharmaceutical compositions for the production of anti-cancer vaccines.

20 cl, 6 dwg, 1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and represents a method of obtaining useful metabolites with the application of bacteria of the Enterobacteriaceae family, in particular bacteria, belonging to the genus Escherichia, which is modified in such a way that it contains a genetic expression system, including a transcription apparatus, regulated by a protein of the LysR type, and modified in such a way that the self-induced positive regulation of the feedback type in the said system is mediated by a coinducer.

EFFECT: method is suitable for the production of L-amino acids with a branched chain, in particular L-valine, L-isoleucine and L-leucine, higher alcohols and D-pantothenic acid; invention makes it possible to obtain L-amino acids with the high degree of efficiency.

23 cl, 2 dwg, 5 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: proposed RNA-aptamer is a 57-unit mixed-type oligonucleotide having the nucleotide sequence GGGAGGACGAUGCGGUGUUUUCUGAGUACAUCUCUGCCCCACCCUU GUUUACCCCCA, where A, G are ribonucleotides, U, C are 2'-desoxy-2'-fluoro-ribonucleotides, has the ability to recognise autoantibodies specific to disseminated sclerosis.

EFFECT: characterised invention binds specifically and highly affine with autoantibodies specific to disseminated sclerosis, and can be used for the diagnostics of disseminated sclerosis.

3 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and can be used to determine a human genotype by polymorphism in matrix metalloproteinase MMP9-1562 C>T (rs3918242) gene. The method is based on the establishment of a melting profile with fluorescence-labelled specific oligonucleotide samples. The method uses an allele-shared pair of primers, fluorescence-labelled allele-specific oligonucleotide samples different for each allele and a general oligonucleotide labelled with a fluorescence extinguisher of the following nucleotide composition: MMP9-1562s CGAAACCAGCCTGGTCAACG; MMP9-1562a TCTGCCTCCCGGGTTCAAGC; MMP9-1562p1 GGCGCACGCCTATAA-FAM; MMP9-1562p2 GGCGCATGCCTATAA-HEX; MMP9-1562pq BHQ1-ACCAGCTACTCGGGAGGC-3'-(P), wherein FAM means the fluorescence extinguisher FAM, HEX means the fluorescence extinguisher HEX, BHQ1 means the dark fluorescence extinguisher attached to 5'-terminal nucleotide. Referring the sample to a homozygote or a heterozygote by the allele is determined by a DNA melting profile shape that is a maximum of the first fluorescence curve derivative.

EFFECT: invention enables providing more reliable and accessible genotyping.

1 dwg

FIELD: biotechnology.

SUBSTANCE: invention provides method of simultaneous (multiplex) amplification and fluorescent marking of DNA of several segments of the genome of mycobacteria of tuberculosis complex (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum and M. microti). Then the hybridisation or electrophoretic analysis of the sequences of these segments is carried out. The multiplex PCR is carried out in a single reaction volume with use of specific and adapter primers, the fluorescent substrate embedded into the growing DNA chain during PCR, and genomic DNA as a matrix. The multiplex PCR comprises two consecutive amplification profiles, different in annealing temperatures of specific and adapter primers by not less than 10°C. The invention enables to carry out the analysis of sequences of these genes to identify mutations associated with resistance to anti-tuberculosis preparations.

EFFECT: method according to this invention reduces the number of stages of amplifications to obtain single stranded fluorescent-labelled PCR-products, eliminates the transfer of a DNA-matrix from one reaction volume to another, which increases the stability of the procedure to contamination with PCR-products and reduces significantly the time and complexity of the analysis.

4 dwg, 1 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents pCFP10-DBD recombinant plasmide consisting of an artificial bacterial operon of a chimeric protein including a promoter region of early promoter of T5 bacteriophage, a chimeric protein gene consisting of a sequence of protein antigene CFP10 from Mycobacterium tuberculosis, which is merged with a sequence of a dextrane-binding domain (DBD) of dextrane sucrase Leuconostoc citreum KM20, and a transcription terminator, a bacterial operon of beta-lactamase and a bacterial initiation section of replication of ColE1 type. Besides, strain Escherichia coli - a producer of CFP10-DBD chimeric protein is presented. The invention describes a method of immobilisation, concentration and cleaning of the obtained protein on cellulose. The invention describes an immunogenic composition containing CFP10-DBD recombinant protein, which is aimed at induction of immunity against tuberculous infection.

EFFECT: invention enlarges the range of devices used for treatment of tuberculosis.

4 cl, 6 dwg, 1 tbl, 4 ex

FIELD: process engineering.

SUBSTANCE: set of invention relates to biology, particularly, to automated purification of biological specimens at extraction of target matters involving the magnetic field application. Magnetic field application element and heater are integrated for up and down movement. Automatic device comprises unit 100 displacing vertically and horizontally to accommodate pipettes 141, 142 for suction and discharge of fluid. Heater 810 heats a particular unit of holes of multihole tray 420, 420' while element 700 for magnetic field application is arranged on support plate 400 including magnet seat 710. Element 700 accommodates magnet 711 at bottom side of said unit of said tray Proposed method exploits above described device.

EFFECT: higher process efficiency.

26 cl, 48 dwg

FIELD: biotechnology.

SUBSTANCE: bionanoconjugate comprises a nano-sized superparamagnetic particle of cobalt ferrite spinel CoxFe3-xO4, where 0.6≤x≤0.98, obtained by mechanochemical synthesis. To isolate the nucleic acid containing oligo- or poly-A/dA sequence the synthetic single stranded oligonucleotide 5'-dGndTm is used, where n=5-30, m=10-35, one portion of which, consisting of guanine nucleotides in the presence of phosphate anions in the solution is specifically associated with the surface of the nanoparticle and the other - consisting of thymine nucleotides is able to enter into hybridisation with oligo- or poly-A/dA nucleotide sequences. For isolation from the solution in the presence of phosphate anions of specific hetero-nucleotide sequence additionally create a molecule of oligonucleotide-adapter containing the sequence of oligo-dAx at the 3'-end, hybridised with thymine nucleotides of a complex 5'-dGndTm, where n=5-30, m=10-35, and a portion at the 5'-end complementary to a specific hetero-nucleotide sequence in the solution.

EFFECT: invention enables to detect effectively and to isolate the single-stranded nucleic acids.

3 cl, 8 dwg, 1 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry. There are presented pharmaceutical compositions having a concatemer molecule and a kit, as well as using for preparing an agent for immune system modulation or for human's or animal's immune system activity modulation, and a composition according to the invention. The given invention can find further application as an immunomodulatory agent in therapy of various diseases.

EFFECT: what is presented is a concatemer molecule of non-coding nucleic acid containing at least four single-strand sites with non-methylated CG motives for human's or animal's immune system activity modulation.

20 cl, 4 dwg

Up!