The lecture covers the following topics: Manipulation of DNA, RNA and proteins - types of cell cultures, cell fractionation, isolation of proteins and nucleic acids. DNA sequencing. Determination of genomic sequences of the whole organism. Methods of study of protein-protein and protein-DNA interactions. Gene cloning tools - restriction endonucleases, DNA polymerases, ligase and reverse transcriptase, plasmids and cloning vectors. PCR techniques: principle, optimization, types and usage. Mutagenesis in vitro, targeted changes in genetic material, random mutagenesis, mutagenesis by mutagenic oligonucleotides, mutagenesis locally targeted, cluster mutagenesis, use of suppressor tRNA. Creation and use of cDNA and genomic libraries. Gene chips, DNA microarrays, Affymetrix technology. Cloning strategy: Optimization of expression of cloned genes, factors influencing gene expression in foreign hosts, selection markers. Gene cloning in prokaryotes. Cloning of genes in yeast and its use for analysis of the eukaryotic genome. Methods of transfer of foreign genes to eukaryotic cells (microinjection, electroporation, transfection, vector systems, biolistic methods). General characteristics of vectors for the transfer of genes to eukaryotes, selective markers. Genetic manipulation, genome editing techniques, Zn-finger nucleases, TALENs, CRISPR/Cas9. Methods of study of expression and function of genes. Visualization of cells and cell structures - light and fluorescent microscopy, visualization of components in living cells, observation of cell dynamics, electron microscopy - types and applications, particle reconstruction and tomography. Cloning of genes in plants and its use. Gene transfer using vectors derived from Ti-plasmid. RNAi, gene silencing and gene "knock out". Gene cloning in animal cells. Transfer of foreign genes to germ cells (eggs, embryos) of insects, amphibians and mammals. Use of methods of recombinant DNA in agriculture, industry and medicine. Bt-maize, DNA vaccination and "plantibiotics", biocontrol of the environment, protection against bioterrorism, etc. Transgenic animals: Dolly and Polly, Cumulina, human proteins, cloning of the human genome. Gene therapy, the main strategy of gene therapy in vitro and in vivo. Cloning and stem cells. Preparation of transgenic organisms. Risks of preparation of transgenic organisms, rules of safety of work with transgenic organisms. Ethical problems related to interspecies gene transfer and preparation of transgenic organisms. GMO legislation.
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Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, Walter P. (2017). Molecular Biology of the Cell, 6th edition. Garland Science.
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Green M.R., Sambrook J. (2012). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press.
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