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Information × Registration Number 0210U004399, 0107U001637 , R & D reports Title Design of molecular test-systems for detection of drug-resistant mycobacteria and study of uncanonical structures in genome of mycobacteria popup.stage_title Head Limanskii Alexander, Registration Date 20-09-2010 Organization The Mechnikov Research Institute of microbiology and immunology at Kharciv popup.description2 Object of the research – genomic DNA of isoniazid-resistant mycobacteria tuberculosis, pGEMEX supercoiled DNA, genome of mycobacteria tuberculosis and retroviruses, T7 RNA polymerase, DNA – RNA polymerase complexes. Aim – the design of scheme and PCR-based molecular genetic test-systems to detect antituberculosis drug isoniazid-resistant mycobacteria tuberculosis and determinating distribution of uncanonical structures like as potential hairpin-loop structures and triplexes in the genome of the retroviruses as model systems. Methods – polymerase chain reaction, computer analysis, atomic force microscopy. Molecular technologies for differentiation of wild type and mutant mycobacteria tuberculosis by standard PCR. Eight primer sets for molecular genetic detection of mutations and fragments of katG gene mycobacteria tuberculosis by multiplex PCR were designed. Mentioned above sets permit to amplify: 1)complete katG gene; 2)five types of mutations in codon 315 and point mutation in codon 463 of katG gene. Besides that, two primer sets as positive control for amplification one of which contains codon 315 were developed. Perfect interstranded triplexes that can be potentially formed in proviral DNA of widely spread bovine retroviruses like as leukemia virus (BLV) and immunodeficiency virus (BIV). Genome of those viruses was considered as a model of more complicated bacterial one. Inverted repeats that can form hairpin-loop and cruciform structures in the genome of BLV and BIV were determined and maps of hairpins localization were obtained. Retroviruses are shown to be characterized by different qualitative and quantitative composition of hairpin-loop structures. Experimentally existence of hairpins and triplexes was confirmed by visualization of pUC8 supercoiled DNA with cruciform structure and pGEMEX supercoiled DNA by atomic force microscopy (AFM). Complexes of T7 bacteriophage RNA polymerase (RNAP) and DNA template during transcription were visualized by AFM. Images of stable specific complexes between T7 RNAP and terminal fragments of DNA template were captured for single molecules. Range of application: microbiology, molecular biology, nanotechnology. Results of this research can be realized in the Research Institutes of NAS of Ukraine, Academy of Medical Sciences, Academy of Agrarian Sciences and in the system of antituberculosis dispensaries. Significance of the work: developed schemes and molecular genetic sets on the basis of different modifications of polymerase chain reaction permit to increase determination of drug-resistance of mycobacteria tuberculosis for аntituberculosis drug isoniazid from 1-3 months by traditional bacteriological methods up to 1-3 days by PCR. Product Description popup.authors Лиманська Ольга Юріївна Лиманський Олександр Петрович popup.nrat_date 2020-04-02 Close