LABORATORY OF ADVANCED DNA, RNA AND PROTEIN ANALYSIS

Second cycle degree in BIOTECHNOLOGIES FOR FOOD SCIENCE (Ord. 2017)

Campus: LEGNARO (PD)

Language: English

Teaching period: Second Semester

Lecturer: BARBARA CARDAZZO

Number of ECTS credits allocated: 9


Syllabus
Prerequisites: Theoretical knowledge: principles of Biochemistry (academic level), with particular focus on protein Biochemistry. Basis of molecular genetics and molecular biology methods.
Practical skills: basic skills for a laboratory of Biochemistry and Molecular Biology (e.g., knowledge of safety rules, use of micro-pipettes, preparation of solutions).
Examination methods: Written dissertation on the theoretical and practical contents of the course, with particular emphasis on protein 2D-electrophoresis and related techniques for the identification of biological markers. In some instance, a PowerPoint presentation of a scientific paper will also be envisaged.
Course unit contents: 1. Proteomics
Recollections of protein biochemistry. Protein structure and structure-function relationship, forces that drive protein folding and protein-protein interactions, differences between hydro-soluble and membrane proteins.
Definition and goals of proteomics. Properties of the proteome; identification of biological markers by proteomic analyses; advantages and disadvantages of the proteomic approach in comparison with other techniques. Overview on the most common methodologies in proteomics.
Theoretical and practical principles of two-dimension gel electrophoresis (2D-electrophoresis). General principles of protein electrophoresis; isoelectric point and isoelectrofocusing; SDS-PAGE.
Techniques for protein extraction, solubilization and purification. Extraction of proteins from the biologic milieu; protein denaturation; preparation of the sample for the 2D-electrophoresis; reduction and alkylation.
Protein identification. Gel staining; tryptic digestion; liquid phase chromatography and mass spectrometry (hints); peptide fingerprinting and sequencing; database mining. Immuno-based techniques for protein identification.
Other techniques in proteomics. The ETTAN-DIGE® technique for 2D-electrophoresis. Techniques for membrane protein identification. Protein microarrays and antibody microarrays. Recent mass spectrometry techniques in proteomics (shotgun proteomics).
Lab sessions: By exploiting principles and techniques learned in the first part of the course, each student will accomplish a complete experiment of 2D-electrophoresis, from protein extraction to gel staining.

2. Molecular biology
Introduction to Molecular Biology methods and application to food analysis.

Description of Methods: Extraction of nucleic acids, PCR and real-time PCR: theoretical principles and application, sequencing methods (Sanger, Next generation sequencing, RNA seq).

Molecular Biology lab session: Molecular typing of bacterial isolates, application of real-time PCR. Next-Generation Sequencing technologies.