Genetic Engineering - MB140P36
Title: Genové inženýrství
Czech title: Genové inženýrství
Guaranteed by: Department of Genetics and Microbiology (31-140)
Faculty: Faculty of Science
Actual: from 2019
Semester: winter
E-Credits: 6
Examination process: winter s.:combined
Hours per week, examination: winter s.:3/2, C+Ex [HT]
Capacity: unlimited
Min. number of students: unlimited
4EU+: no
Virtual mobility / capacity: no
State of the course: taught
Language: Czech
Level: basic
Note: enabled for web enrollment
Guarantor: Mgr. Václav Vopálenský, Ph.D.
Teacher(s): Mgr. Václav Vopálenský, Ph.D.
Co-requisite : {At least one of the following subjects ((MB140P71, MB140P21, MB140P41)}
Opinion survey results   Examination dates   WS schedule   
Annotation -
The lecture is focused on description of basic methods and strategies exploited in genetic engineering of both caryotic and aucaryotic organisms in vitro, and in vivo. The main groups of methods are isolation of DNA or RNA, chemical and biochemical synthesis of oligonucleotides and polynucleotides, construction of large ds DNA in vitro, degradation and modification of DNA, reverse, transcription, amplification of DNA by PCR, recombination of DNA in vitro, DNA transformation of cells with isolated DNA, analysis of recombinant DNA and selection of recombinant molecules, viruses, cells or multicellular organisms. The main strategies are following: Selection strategies, site-specific and random mutagenesis, deletion mutagenesis, cloning, preparation and exploitation of DNA or c-DNA libraries, expression of foreign genes, constructions of various types of vectors, sequencing and genetic manipulation in vivo. The methods and strategies are demonstrated mostly on E. coli and S. cerevisiae.
Last update: Vopálenský Václav, Mgr., Ph.D. (28.08.2017)
Literature -

·      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)

Last update: Vopálenský Václav, Mgr., Ph.D. (28.08.2017)
Requirements to the exam -

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.

Last update: Vopálenský Václav, Mgr., Ph.D. (25.10.2019)
Syllabus -

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

Last update: Vopálenský Václav, Mgr., Ph.D. (25.10.2019)