The main goal of the course is to introduce and familiarize students with the fundamental laboratory techniques providing a basic understanding of the methodologies employed for the analysis of nucleic acids and proteins. The course will focus on DNA, RNA and proteins purification and characterization, in vivo and in vitro (PCR) molecular cloning and DNA sequencing. The course will also cover the major principles of enzymatic assays and basic cell culture techniques, including genetic engineering of animal cells. The teaching on the course is split between formal lectures and tutorials most of which are held in dedicated teaching laboratories. This laboratory-based work will provide hands-on experience with nucleic acid and protein isolation, quantification, and characterization. Laboratory activities will include spectroscopy methods (UV-visible), gel electrophoresis (SDS-PAGE, agarose), basic molecular and cell biology techniques such as recombinant DNA, PCR, restriction enzymes, plasmid DNA, sterile techniques, growing bacteria and animal cells, cloning, protein and enzyme assays. Specific goals of the laboratory work is to provide students with the practical skills needed in the basic techniques used in biochemistry, cell and molecular biology, and to let them acquire the knowledge to correctly analyze the experimental results.
At the completion of this course, students are expected to have an understanding of the major concepts and theoretical principles of basic techniques used in molecular and cell biology as well as the basic practical skills needed to use the fundamental laboratory methodologies.
Cellular experimental systems: methods in bacterial and animal cell cultures (principles, techniques, and applications). Cell-based DNA cloning: principles of cell-based molecular cloning, use of restriction endonuclease enzymes, ligation, cloning vectors, transformation, screening of colonies. Methods for gene cloning and expression: factors affecting gene expression, expression vectors , cellular systems for gene expression (bacteria , yeast, mammalian cells ), markers and signals, mutagenesis. Gene knockdown methods in cell systems: gene silencing by small interfering RNA (siRNA), microRNA (miRNA), morpholino oligonucleotides.
Modulo: Metodologie molecolari
Fundamental laboratory techniques: overview and principles of centrifugation, chromatography, electrophoresis and spectrophotometry. Protein purification: introduction and basic principles; protein quantification methods to determine protein concentration. Analysis of proteins by gel- electrophoresis and blotting. Methods for determination of enzyme activity: general principles of enzymatic assays. Extraction, purification and quantification of nucleic acids. Analysis of nucleic acids by gel-electrophoresis and blotting. cDNA and genomic libraries, library screening methods. Molecular cloning using polymerase chain reaction (PCR) technology: principles of the PCR method, optimization, primer design, troubleshooting; cloning of PCR products, types of PCR (Long-Range PCR, RT-PCR, quantitative PCR or 'Real-Time'), applications of PCR. Analysis of gene expression: Northern blot, RT-PCR, in situ hybridization, reporter genes, promoters, DNA-binding proteins, transcriptome analysis, RNA-Seq.