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Last update: Mgr. Václav Vopálenský, Ph.D. (28.08.2017)
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Last update: Mgr. Václav Vopálenský, Ph.D. (28.08.2017)
· Sambrook, J., Fritsch, E.F., Maniatis, T., (1989), Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press · J. Sambrook, D. Russell - Molecular Cloning: A Laboratory Manual (Third Edition / Fourth Edition, CSHL Press) · Vondrejs V. (1997, 2001, 2003, 2011), Genové inženýrství I-IV, Karolinum · (V. Vondrejs; Otazníky kolem genového inženýrství, Academia 2010) · Brown T.A. (2006, 2010) Gene Cloning and DNA Analysis: An Introduction; Blackwell Publishing Incorporated · T. Nicholl - An Introduction to Genetic Engineering, Third Edition (2008) · S.B. Primrose – Principle of gene manipulation and genomics, Seventh Ed. (2006) |
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Last update: Mgr. Václav Vopálenský, Ph.D. (25.10.2019)
The exam is usually written. It is necessary to write an answer to the questions (about 10 in total), so it is not a simple ticking of answers. Test timeout is 120 minutes. Scoring of answers is based on the difficulty of the questions (4-3-3-2-2-2-1-1-1-1 points can be obtained for individual questions, ie 20 points in total). The classification is as follows: 18-20 points = excellent; 15-17.5 points = very good; 12-14.5 points = good; less than 12 points = failed. |
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Last update: Mgr. Václav Vopálenský, Ph.D. (25.10.2019)
Lecture 1 - Introduction to the Genetic Engineering (GE); advantages x disadvantages of GE, basic principles of GE, history of GE (1953-2019), horizontal gene transfer (basics of conjugation, transduction, transformation, induced protoplast fusion Lecture 2 - Purification of nucleic acids (NA); cell lysis, purification/concentration of nucleic acid, isolation of plasmid DNA, RNA isolation Lecture 3 - Separation of nucleic acids; DNA/RNA electrophoresis, purification of DNA fragments from agarose gels, spectrophotometric quantitation of NA, chemical synthesis of oligonucleotides, biochemical synthesis of DNA Lecture 4 – PCR; history of the PCR, components of PCR reaction mix, thermal profile of PCR , PCR inhibitors, PCR optimization, PCR variants Lecture 5 - RT-PCR; DNA fragmentation; mechanical fragmentation of DNA, sequence non-specific degradation of DNA, sequence specific degradation of DNA, nicking / homing endonucleases Lecture 6 - Modification of DNA ends; nucleases an polymerases and mode of their action, labelling of DNA ends using Klenow fragment and T4 DNA polymerase, working with DNA linkers and adaptors, labelling of NA Lecture 7 - DNA cloning; ligation history, ligases, cloning of cohesive and of blunt ends, vectors, features of vectors Lecture 8 - Transfection of DNA into organisms, electroporation, lipofection, transient x stable transformation (selectable markers); Mutagenesis - chemical mutagens, mutagenic PCR oligonucleotide-directed mutagenesis; sequencing - Maxam-Gilbert and Sanger sequencing, pyrosequencing Lecture 9 - RNA transcription, Protein expression systems - of E. coli based expression system, problems with expression in E.coli, in vitro translation systems, couplet in vitro transcription/translation, laboratory strains of E. coli used in GI Lecture 10 - Genetic manipulations in eukaryotes (mainly in Saccharomyces cerevisiae (S.c.)), vectors, yeast transformation, homologous recombination in yeast, mutagenesis in vivo, expression of a foreign gene in yeast, expression of a foreign gene in mammals cell lines, vectors and promoters used for expression Lecture 11 - Protein interaction technologies; the two hybrid screen, co-immunoprecipitation, GST pull-down assays; Preparation of genomic and cDNA libraries Lecture 12 - Genome modification in vivo - Zinc Finger Nucleases, TALEN, CRISPR/Cas9 Lecture 13 - Analyses of gene expression, Northern blotting, mapping RNA with nuclease I, mapping-protein-binding sites by DNAseI foot-printing, gel retardation assays, gene therapy |