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Lecturer(s)
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Šebela Marek, prof. Mgr. Dr.
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Frébort Ivo, prof. RNDr. CSc., Ph.D.
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Course content
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Enzymes used in molecular biology: DNA and RNA polymerases, ligases, nucleases, phosphatases. Hybridization techniques and ways how to prepare hybridization probes. Cloning strategies and cloning vectors: plasmids, bacteriophage, BAC, selection markers, TA-cloning, TA TOPO cloning, Golden Gate cloning, recombination cloning - GATEWAY, yeast recombinant cloning. Heterologous expression in microorganisms. Regulation of expression under the control of T7 promoter. Yeast expression systems - Saccharomyces, Pichia. Polymerase chain reaction - principle, types of PCR reactions - RACE PCR, RLM RACE PCR, inverse PCR, in situ PCR, multiplex PCR, TAIL PCR. Quantitative real-time PCR - principle and evaluation. Mutagenesis - type of mutations and mutagens, genetic mapping, site-directed mutagenesis. Genome editing - insertional libraries, mouse knock-out model. Tools for genome editing - zinc-finger nucleases, TALEN, CRISPR/Cas9. Sequencing, Sanger method, massive parallel sequencing methods of second and third generation, pyrosequencing, reversible cyclic termination, SMRT (single molecule real-time) sequencing. Sequencing principles of following companies: Illumina, Roche, PacBio, SOLiD, Ion Torrent, Oxford Nanopores. Transgenic plants, principle of plant cell transformation, types of selection. Usage guidelines - Bt plants, round up ready cultivars, etc. Plants as a source of therapeutic protein and compound production - edible vaccines, plantibodies. Transfection of animal cells - methods. Manipulation with stem cells. Transgenic animals - utilization and preparation. Xenotransplantation. Cell cloning - therapeutic cloning. Gene therapies, viral vectors for human cell transformation. Alternative ways for recombinant DNA delivery to somatic cells. Gene therapy for monogenic diseases and polygenic diseases. Examples of successful gene therapies.
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Learning activities and teaching methods
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Dialogic Lecture (Discussion, Dialog, Brainstorming)
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Learning outcomes
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The subject covers the following topics: tools of molecular cloning - restriction endonucleases, DNA polymerases, ligases, reverse transcriptases, genome editing nucleases, plasmids and cloning vectors; PCR techniques - principle and optimization, different types and utilization; cloning strategies: optimization of cloning gene expression, selection markers; transgene transfer into eukaryotic cells; gene silencing and gene knock-out. Preparation and practical utilization of transgenic plants and animals; therapeutic cloning and gene therapy.
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Prerequisites
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Successful graduated studium of Bachelor of Science and Magister of Science in Biochemistry.
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Assessment methods and criteria
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Oral exam
Oral examination - successfuly answered three questions of general knowledge.
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Recommended literature
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Alberts, B. Molecular Biology of Cell, 6th edition, Garland Publishing (2015)..
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Brown T.A. (2008). Klonování genů a analýza DNA. Nakladatelství UP Olomouc.
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Glick B.R., Pasternak J.J., Patten Ch. (2010). Molecular Biotechnology: Principles and Applications of Recombinant DNA, 4th Edition. ASM Press, USA, 2010. ISBN 978-1-55581-498-4.. Washington DC.
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M.R. Green a J. Sambrook. (2012). Molecular Cloning. A laboratory manual 4th edition, Cold Spring Harbor Laboratory Press, New York,.
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Snustad, D. P., Simmons, M. J. (2017). Genetika. Brno: Munipress.
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