Syllabus of lectures: 1. Basic terms used in genetic engineering. 2. Replication of DNA. 3. Preparation of effective expression systems. Preparation of recombinant DNA. 4. Construction of „gene library“. 5. Selection of required clones. 6. Mutagenesis in vitro. 7. Mobile genes, translocable elements. Use of viruses, bacteriophages. 8. Construction of recombinant DNA for procaryotic or eucaryotic systems. 9. Transfer of genes to procaryotic or eucaryotic cells. 10. Diagnostic methods based on molecular cloning, gene therapy. 11. Genetically modified organisms – EU legislation. 12. Commercial use of molecular cloning, prospective, protection. Biotechnologies. 13. Computer techniques in molecular cloning. Syllabus of practical lessons: 1. Purification of plasmid DNA a) analytical, b) preparative, c) determination of DNA concentration 2. Purification of chromosomal DNA a) analytical, b) preparative, c) differentiation of plasmid and chromosomal DNA. 3. Electrophoretic differentiation and visualisation of DNA a) preparation of agarose gels, b) preparation of polyacrylamide gels, c) visualisation of DNA. 4. Use of restriction endonucleases a) complete splitting by restriction endonucleases, b) partial splitting by restriction endonucleases, c) construction of simple physical maps. 5. Isolation of genes from plasmid or chromosomal DNA a) methods of separation of DNA genes from agarose gel, b) purification of isolated genes, c) amplification of isolated fragment by PCR. 6. Use of vectors in preparation of recombinant DNA a) for prokaryotic systems b) for eukaryotic systems c) amplification of eukaryotic DNA in prokaryotic cells d) inducible or dependent vectors 7. Preparation of recombinant DNA – genetically modified organisms a) preparation of suitable vector system, b) ligation, c) transformation 8. Amplification of recombinant DNA in vivo a) construction of „gene library“, b) preparation of „competent cells“, c) transformation. 9. Selection of required clones a) selection on the basis of antibiotic-resistance, b) selection on the basis of other phenotype properties, c) restriction – endonuclease analysis of recombinant DNA. 10. Methods of testing the products of recombinant DNA a) in vivo, b) in vitro, c) mutation, d) utilization of transpozones. 11. Transfer of genetic information into the eukaryotic system a) work with cell line, b) transformation into the eukaryotic system, c) selection methods. 12. DNA diagnostic methods a) PCR, b) hybridization, c) non-radioactive labeling of DNA, Southern blotting. d) Inactivation of functional genes e) Genetically modified micro-organisms. f) Test. 13. Evaluation of student’s activity - Giving credits. Question areas for examination: 1. Principles of preparation of recombinant DNA: Isolation and purification of nucleic acids, separation of nucleic acids (types of electrophoresis, PFGE, 2D electrophoresis); construction of gene libraries, principles of gene isolation. 2. Enzymes in recombinant DNA techniques: Restriction endonucleases, methylases, DNA and RNA polymerases, reverse transcriptases, ligases, kinases, phosphatases, proteases; Principles of physical mapping of nucleic acids. Regulatory signals affecting gene expression. 3. Selection and analysis of prepared clones: Selection and analysis of clones (physical analysis, functional detection, hybridization and serological methods). 4. Vectors: Types of cloning vectors – their properties and use, expression systems; recipient strains, methods of „insertion of genetic information“ into host cells. 5. Mutagenesis In vitro: methods and their practical use. 6. Hybridization methods: Radioactive and non-radioactive labeling; types of hybridization methods. 7. DNA diagnostic methods: PCR – principle, types, conditions, designing primers, technique variations. 8. Sequencing of nucleic acids: Methods of DNA sequencing (Sanger, Maxam-Gilbert). 9. Genetically modified organisms: Preparation, use, safety, legislation. 10. Biotechnological processes: Division of biotechnologies, their use. |
