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
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.