Syllabus
Genome manipulations and reverse genetics
Selection and reporter genes, tools for genome manipulation (site-specific recombinases, site-specific endonucleases, homologous recombination) reverse genetics - random gene inactivation (transposon mutagenesis, TILLING, gene trap), targeted gene inactivation (knock-out, RNA-interference), regulated gene expression.
Genomic modifications in model organisms
Characteristics and specifics of model organisms of molecular biology (E. coli, S. cerevisia, D. melanogaster, C. elegans, A. thaliana, mouse, human - tissue culture), creation of knock-out, conditional knock-out, knock-in, transformation methods, vectors.
Human gene modifications - gene therapy
In vivo and ex vivo gene therapy, stem cells, cloning, induced pluripotent cells, viral vectors for gene therapy (retroviral, adenoviral, AAV), their production, non-viral vectors (transposon), transfection methods, polymer carrier DNA design, gene therapy approaches (expression of functional allele of gene, expression of suicide gene, decrease of gene expression, repair of damaged gene), antisense therapy.
DNA sequencing
Chronological view of sequencing methods (chemical degradation method, dideoxy method, pyrosequencing, second generation methods (454, Illumina, Ion Torrent), third generation methods (PacBio), de novo vs. re-sequencing, exome sequencing, genome sequencing, genomic projects .
Methods of gene expression studies
Methods of study of individual gene expression: EST, Northern blot, qRT-PCR (PCR kinetics, Ct value, probes for detection, normalization, PCR efficiency).
Methods of study of transcriptome: DNA chips (principle, presentation of results: temperature maps, clustering; particular areas of use of DNA chips), RNA sequencing (RNA-Seq).
Protein study methods
Protein concentration determination, separation: PAGE (denaturing, native), staining, protein isolation and purification using affinity tags, immunolabeling-antibodies (primary, secondary, detection, production), immunoprecipitation,
Western blot, ELISA, methods of determination of protein-protein interactions at the level of individual proteins (pull-down assay, proximity ligation assay) and whole proteome (coimmunoprecipulation, TAP, 2-hybrid system, complement assay, phage display), degron, protein arrays ( analytical vs. functional chip).
Proteomics mass spectrometry: separation, ionization techniques, types of analyzers, tandem MS.
Fluorescence and fluorescent labeling
Fundamentals of fluorescence, filters and light sources, fluorescent labeling of cells and organelles, use of fluorescent probes, fluorescent probes for measuring intracellular pH, ion concentration, membrane potential.
Fluorescence microscope, confocal microscope and two-photon confocal microscope. Green fluorescent protein (GFP) and its variants. Possibilities of using fluorescent proteins, FRET, FRAP, FLIM.
Flow cytometry. flow cytometer and cell sorter, principle of measurement, dot plot, population gateway, multicolour flow cytometry, mass cytometry.
Advanced microscopic techniques
Atomic force microscopy, near field microscopy, TIRM, high resolution fluorescence microscopy (4Pi, STED, PALM / STORM).
Methods of the future
Third-generation DNA sequencing (nanopore sequencing and other techniques developed), single-cell techniques (laser micropreparation, optical tweezers, magnetic tweezers), DNA origami and DNA nanoarray, lab-on-chip microfluidic chips.
Annotation
The class reviews current methods used in molecular and cellular biology. The topics cover a broad spectrum of methods from genome editing methods, nucleic acids sequencing and manipulation, protein detection and identification, use of fluorescent probes and proteins to modern microscopic techniques.
Basic understanding of molecular and cellular biology and genetic engeneering is required.