1) History of gene cloning. Structure of the genomes of prokaryotic and eukaryotic organisms. Genetic manipulations. Use of the bacterium Escherichia coli in genetic engineering. 2) Vectors used for gene cloning: plasmids and bacteriophages. Isolation of total cellular DNA from prokaryotic and eukaryotic organisms. Isolation of plasmid and bacteriophage DNA. Determination of DNA concentration and assessment of the quality of isolated DNA. 3) Enzymes used for DNA manipulation: nucleases, ligases. 4) Polymerases, modification enzymes, topoisomerases. Cloning vectors for E. coli: vectors based on E. coli plasmids. Transfer of plasmid DNA into bacterial cells. 5) Vectors based on lambda bacteriophage, vectors based on gateway technology, cosmids, vectors based on M13 bacteriophage, phagemids. Transfer of phage DNA into bacterial cells. Cloning vectors for eukaryotic organisms: vectors for yeasts and other fungi, vectors for animals. Microinjection. 6) Plant vectors. Transformation of plant cells: transient and stable transformation, direct and indirect transformation methods. Acquisition of a clone of the studied gene: direct selection, marker rescue, genomic libraries, cDNA libraries, expression libraries, and methods used for library screening. 7) Strategies for determining gene location on small and large DNA molecules. Study of gene structure. Analysis of gene expression. 8) Genome sequencing: sequencing methods, whole-genome shotgun sequencing, clone-by-clone sequencing. Identification of genes in sequenced genomes. Transcriptome analysis using DNA microarrays and RNA sequencing (RNA-Seq). Basic methods used for proteome analysis. 9) Gene cloning and DNA analysis in medicine: production of recombinant pharmaceuticals, identification of genes responsible for human disorders, gene therapy. 10) Gene cloning and DNA analysis in agriculture: gene addition strategies in plant genetic engineering, gene silencing using antisense RNA and RNA interference, genome editing using programmable nucleases, risks associated with the cultivation of genetically modified crops. 11) DNA analysis in forensic genetics: identification of crime suspects, kinship studies, sex determination. Archaeogenetics.
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