[DNA Fragment Enrichment for High-Throughput Sequencing].

This review describes the application of oligonucleotides, which are mainly obtained using DNA synthesizers of a new generation (microarray DNA synthesizers), for the enrichment of target genomic fragments. The methods of molecular hybridization, polymerase chain reaction, and CRISPR-Cas9 system for this purpose are considered. Examples of the practical use of the developed methods for research and diagnostic purposes are given.

[Application of Array-Based Oligonucleotides for Synthesis of Genetic Designs].

The application of array-based oligonucleotides in biological studies is described. These oligonucleotides are mainly used to design large libraries of various nucleotide sequences, which are applied to study protein-nucleic acid interactions, splicing, transcription, translation, and other regulatory processes in mammalian, yeast, and bacterial systems. The application of gene libraries generated by array-based nucleotides along with advanced methods of the combination of DNA duplexes will make it possible to obtain complex genetic designs for synthetic biology.

[Lifestyle and health of students].

The article presents the results of the evaluation of the most significant risk factors related to lifestyle and health of medical students of different courses andfaculties. The obtained data testify that out of the total number of factors that have a significant influence on the formation of bases of student's healthy lifestyle and health, the most typical are the mode of employment, the total workload, material well-being, living conditions of the majority of today's students, as well as the conditions of nutrition, physical activity, the presence or absence of such factors as smoking, frequency of consumption of alcoholic beverages.

[On-microchip PCR for detection of influenza A viruses subtypes, circulating in the human population].

A oligonucleotide microchip was developed for revealing Influenza A viruses subtypes, circulating in human population: pandemic H1N1 swine influenza viruses, seasonal H1N1, H2N2, H3N2, H5N1, H9N2, H7N9. Typing of influenza virus was performed by on-microchip PCR. We used immobilized primers-probes selected for the neuraminidase gene that allows determining both subtype of neuraminidase and subtype of hemagglutinin.

[A compact microarray for sub-typing of influenza A virus].

A universal oligonucleotide hybridazation microchip 6 x 5 spot (4 x 4 mm) for influenza A virus subtyping was suggested, functioning on a principle one spot--one subtype. This microchip with additional printing quality control is a prototype of a biosensor for detection of influenza A virus and typing of 15 subtypes of hemagglutinin and 9 subtypes of neuraminidase.

[The second generation universal oligonucleotide microarray for subtyping of influenza virus A].

The microchip for influenza A subtyping was developed, functioning on a principle "one spot--one subtype". Each spot contains the set of oligonucleotide probes, specific for a particular subtype of hemagglutinin, neuraminidase or matrix gene. Reliability of the proposed chip version is the same as for earlier created in our group full-size microchip for separate hemagglutinin and neuraminidase subtyping. To visualize the image, analyzed DNA can be labeled by either fluorescent dye or biotin with the further fixation in system streptavidin-gold nanoparticles and image development by silver precipitation. In the second case common version of scanner can be used for the image analysis, that essentially simplifies procedure of influenza A subtyping.

[Oseltamivir resistance depends on the position 273 amino acid of neuraminidase of the type A influenza virus (H1N1), circulating in human population].

The structure of neuraminidase of the type A influenza virus (H1N1) spreading in the human population was analyzed. The obtained results indicate a significant correlation between the oseltamivir sensitivity and the nature of the amino acid localized not only to neuraminidase position 274, but also to position 273 of this protein. Phenylalanine at position 273 in neuraminidase indicates a higher propensity to influenza virus mutation H274Y, leading to the appearance of resistant strains. It is suggested that the mutation at position 273 may be one of the characteristics allowing type A influenza virus to be ascribed to a pandemic or a seasonal type.

[Microarray for diagnostics of human pathogenic influenza A viruses subtypes].

An oligonucleotide microchip was developed for diagnostics of human pathogenic Influenza A viruses subtypes. It contains discriminating probes for H1-, H2-, H3-, H5-, H7- and H9-subtypes of hemagglutinin and for N1-, N2-, and N7-subtypes of neuraminidase. The additional set of probes was used for M-gene of Influenza A viruses definition. Microchip was tested on samples pathogenic H5N1 avian influenza viruses, pandemic H1N1 swine influenza viruses and seasonal H1N1 and H3N2 influenza viruses. The microchip can be used for the analysis of both cultured strains and clinical specimens.

[Oligonucleotide microarray for subtyping of influenza virus A neuraminidase].

Microarray for influenza A neuraminidase subtyping was presented. Selection of oligoprobes proceeded in two steps. First step included selection of peptides specific for each subtype of neuraminidase. At the second step oligoprobes were calculated using found peptides structures with the subsequent additional selection of the most specific and representative probes. From 19 to 24 probes were used for determination of each subtype of neuraminidase. Microchip testing for 19 samples with the most widespread types (N1 and N2) specifies in unequivocal definition 18 of them and only one isolate has not been identified.

[Subtyping of influenza virus A hemagglutinine with hybridization microarray].

An oligonucleotide microarray for influenza A hemagglutinine subtyping was presented. The number of probes for determination of each subtype hemagglutinine (H1-H13, H15, H16, pandemic flu H1N1)varied from 13 to 28. When testing of the microarray using 40 type A influenza virus isolates the hemagglutinin subtypes were unambiguously determined for 36 specimens.

[Identification of human pathogenic variola and monkeypox viruses by real-time polymerase chain reaction].

A kit of specific oligonucleotide primers and hybridization probes has been proposed to detect orthopoxviruses (OPV) and to discriminate human pathogenic viruses, such as variola virus and monkey virus by real-time polymerase chain reaction (PCR). For real-time PCR, the following pairs of fluorophore and a fluorescence quencher were used: TAMRA-BHQ2 for genus-specific probes and FAM-BHQ1 for species-specific ones (variola virus, monkeypox virus, ectomelia virus). The specificity of this assay was tested on 38 strains of 6 OPV species and it was 100%.

[TaqMan probes based on oligonucleotide-hairpin minor groove ligand conjugates].

Hybridization of TaqMan probes derived from oligonucleotides containing fluorophores (fluorescein, FAM, or tetramethylrhodamine, (Tamra)), fluorescence quenchers (BHQ1 or BHQ2), and a conjugated hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues connected through an aminobutyric acid residue were proposed for discrimination of point mutations using the real time PCR technique. Identification of point A/C substitution was shown to be highly specific for hepatitis C virus subtypes 1a and 1b with two variants of the probe (5'-3'): ATTGAGCGGGTTTAp-BHQ2-MGB for subtype 1a and FAM-ATTGAGCGGGTTGAp-BHQ1-MGB for subtype 1b. Perfect duplexes (A.T and G.C pairs) increase fluorescence in the process of amplification, whereas imperfect duplexes (A.G and T.C pairs) induce no fluorescence changes. This phenomenon enables simultaneous genotyping of hepatitis C virus subtypes 1a and 1b.

[An oligonucleotide microarray for detection and discrimination of orthopoxviruses based on oligonucleotide sequences of two viral genes].

An oligonucleotide microarray for detection and identification of orthopoxviruses was developed. Genus specific and orthopoxvirus species-specific regions of the genes encoding chemokine binding and alpha/beta-interferon binding proteins were used as a target. The developed microarray allows the variola, monkeypox, cowpox, vaccinia, camel-pox and ectromelia (mousepox) viruses to be distinguished with a high degree of reliability.

Reversible covalent attachment of cholesterol to oligodeoxyribonucleotides for studies of the mechanisms of their penetration into eucaryotic cells.

A method in which a cholesterol moiety was covalently attached to oligonucleotides via a disulfide bond has been proposed as a means for studying the penetration of oligonucleotides into living cells. This bond may be cleaved by a mild treatment with thiol-containing reagents during some stages of the uptake process. Attachment of the cholesterol moiety resulted in a 30-50-fold increase in uptake of the oligonucleotide derivative by T24 human carcinoma cells. However, more than 80% of the oligonucleotide derivative remained on the external surface of the cellular membrane. Within the cytoplasm, the oligonucleotide derivatives were found in endosome-like vesicles which were observed during the first 6 h following treatment. Oligonucleotide moieties never cross the membrane, and endocytosis, with or without receptors, is the principal mechanisms for cellular uptake. Only about 15% of the oligonucleotides that penetrated the cells were found in the nuclear fraction. Treatment of the cells with dithiothreitol resulted in a release of most of the cell-associated oligonucleotide derivatives from the external surface of the membrane, but did not change the chemical state or intracellular distribution of the penetrated oligonucleotide derivatives. Mechanisms of the binding of cholesterol-modified oligonucleotides to cellular membranes, non-receptor mediated endocytosis and the role oligonucleotide transportation mechanisms play in determining the fate of penetrated oligonucleotides within the cell are discussed.