Learning objectives
KNOWLEDGE AND UNDERSTANDING
Students will gain theoretical and practical principles at the base of the main methodologies used for the study and analysis of proteins and nucleic acid.
APPLYING KNOWLEDGE AND UNDERSTANDING
The course aims to provide the tools necessary to address the complex molecular analysis of issues of interest in the field of food science.
Prerequisites
Students should have a solid background in Biochemistry.
Course unit content
The course will cover the main methods used in the study and analysis of proteins and nucleic acids.
The following topics are considered:
-Introduction to nucleic acid structure and stability.
-Methods of extraction and purification of nucleic acids.
-Electrophoresis of nucleic acids.
-Enzymes used for DNA manipulation: DNA polymerases, nucleases, ligases. Restriction enzymes and RFLP.
-DNA sequencing: chemical degradation method, chain termination method, pyrosequencing, thermal cycle sequencing, NGS sequencing.
-Qualitative and quantitative PCR (Polymerase chain reactions) and RT-PCR techniques.
-Fingerprinting techniques: RAPD, RFLP, AFLP.
-Cloning of exogenous genes, transformation of host organisms and selection of recombinants. Recombinant proteins production.
-Probe preparation and labelling for Southern e Northern Blotting and Microarray
-Methods used in the preparation of transgenic organisms and recombinant proteins for the agri-food sector. Bt corn and RoundUp ready soybean. Analytical methods for the determination of authorised and non-authorised GMOs in food products. Antisense technology. Cloned animals.
-Protein extraction and purification.
-Differentiated protein precipitation. Protein chromatographic separation techniques: hydrophobic interaction, ion exchange, dimensional exclusion and affinity/immunoaffinity chromatographies.
-Protein electrophoretic separation techniques: polyacrylamide gel electrophoresis, isoelectric focusing and two-dimensional electrophoresis.
-Immunochemical methodologies. Overview of the biochemical basis of immune response and of the structural/functional properties of antibodies. Preparation of polyclonal and monoclonal antibodies. Utilisation of antibodies as analytical reagents: immunoprecipitation, immunodiffusion, immunoelectrophoresis, Western Blotting, immunoenzymatic tests (ELISA).
-Enzymology and its applications. Enzyme mechanism of action: main factors of enzymatic catalysis. Enzyme kinetics in the stationary phase and determination of Kcat, Km and
Kcat/Km. Technological applications of enzymes in food industry: use of beta-galactosidase and thermolysin.
-Enzyme inhibitors, reversible and irreversible inhibitors, competitive and non competitive inhibitors. Preparation of stable enzymes: covalent and non-covalent immobilization. Enzymes from thermophilic microrganisms.
-Biochemical basis of BSE.
Bibliography
Wilson K., Walker J.
Principles and Techniques of Biochemistry and Molecular Biology
Reece J.R.
Genes and Genomes
Teaching methods
Oral lessons with the use of multimedia equipment and classroom exercises.
