Learning outcomes of the course unit
The course will provide the methodological tools to resolve protein and nucleic acid mixtures and to calculate the qualitative and quantitative measurements of specific components in food diagnostics, based on chromatographical and electrophoresis separation techniques, on sequencing techniques for protein and DNA and on various immunological and molecular biology techniques. The molecular biology techniques used to obtain transgenic organisms and recombination proteins in the agri-food sector will also be explained. Ample prominence will be given to enzymes and their analytical applications and technology in the foodstuff sector.
Course contents summary
Methodologies in molecular biology. Summary of the structure of nucleic acids. DNA fusion and renaturalization. Spectroscopic properties of nucleic acids. Activity of the main enzymes involved in duplication and transcription processes. Extraction, purification and quantification of DNA and RNA. Electrophoresis: general principles. Electrophoresis of nucleic acids. Activity of main enzymes used in the manipulation of DNA. DNA sequencing: chemical degradation method, chain termination method, thermal cycle sequencing, use of fluorescent markers for automatic sequencing, pyrosequencing. Principles and applications of the DNA polymerase (PCR) chain reaction. Projecting primers.
Nested PCR, multiplex PCR, reverse-transcription PCR, random amplification of polymorphic DNA (RAPD). Quantitative PCR: real time PCR, principles and applications. Restriction enzymes and RFLP. Cloning of exogenous genes in plasmid carriers by using restriction enzymes, “T/A cloning and “Bolcloning”. Transformation of host organisms and selection for the presence of recombinant plasmids. Methodologies used in the preparation of transgenic organisms of interest in the agri-food sector: Bt maize and RoundUp Ready soya. Use of Agrobacterium tumefaciens. Gene silencing by anti-sense RNA technology. Analysis methods for the presence of authorised and unauthorised GMOs in foodstuffs.
Separation methods used for protein. Summary of the structure of amino acids and protein. Determination of protein concentration. Protein extraction from biological tissue. Differential precipitation of proteins through modifications in pH and ionic force. Dialysis. Ultrafiltration. Chromatographic separation techniques for proteins: hydrophobic interaction chromatography, ionic exchanges, dimensional exclusion and affinity/immuneaffinity . Electrophoresis separation techniques for proteins: denaturing electrophoresis on polyacrylamide gel (SDS-PAGE); native electrophoresis; isoelectric focalization; bidimensional electrophoresis. Revelation methods.
Immunological methodologies. Biochemical basics of the immune response and of the structural/functional properties of antibodies. Preparation of polyclonal and monoclonal antibodies. Use of antibodies as analytical reagents: immunoprecipitation, immunoelectrophoresis, Western Blotting, immunoenzymatic test (ELISA). Biochemical basis of BSE and investigation methods. “Misfolding” pathologies: Bovine spongiform encephalopathy (BSE).
Enzymology and its applications. Main factors in enzymatic catalysts. Enzymatic catalyst mechanisms. Regulation of enzymatic activity. The example of serine proteases. Enzymatic cinetic in a stationary state and determination of kcat, Km and kcat/Km. Enzymatic inhibitors. Reversible and irreversible inhibitors. Competitive and non-competitive inhibitors. Enzyme technology. Technological applications of enzymes in the foodstuff sector: use of beta-galactosidase and synthesis of aspartame. Denaturation and stabilization of enzymes. Preparation of stable enzymes: covalent and non-convalent supporting fixations. Enzymes from thermophilic microorganisms.
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