Corso di Laurea in Biotecnologie

Biochemistry Module:

The purpose of the biochemical methods is to give students the theoretical and practical knowledge of the most important methods of extraction, purification, structural characterization and catalytic proteins, as well as the latest application techniques.

Molecular Biology Module:

The purpose of the Molecular Biology Module is to give students the theoretical and practical knowledge of the most important methods in DNA/RNA sequencing and bioinformatics data analysis

Biochemistry and Molecular Biology Module:

The student should be able to navigate through procedures (and be aware of the limitations of them)

Practical laboratory for the biochemistry method: 16 hours

Practical laboratory for the Molecular Biology module: 16 hours

Biochemistry module

The following techniques will be treated in the Methods in biochemistry module:

• Sample preparation for biochemical analysis, protein compatible buffers and detergents.
• Purification of intracellular and extracellular proteins.
• Protein solubility and precipitation techniques.
• Lambert-beer law and spectrophotometric assays for protein quantitation (Lowry, Bradford, Bicinchonic Acid ) and for enzyme activity.
• Chromatography: IEXC, gel filtration. HPLC, FPLC, gas-cromatography.
• Electrophoresis techniques for purity check. PAGE, IEF and 2D electrophoresis for proteomics.
• Protein blotting and decoration ( avidin-biotin, peroxidase, phosphatase): specific staining for glycoproteins.
• Spectroscopy for protein 2ary and 3ary structure studies (CD, fluorescence, NMR, EPR, AS, IR, Raman): application and basis of structural/functional data obtained.
• Electrochemical techniques: electrodes (pHmeter, reference electrode, Clark electrode). Nernst law and plot: spectroelectrochemical titration. Brief discussion on protein-electrode interactions.
• Lab practicals: Recombinant protein expression systems. purification of a GST-tag fusion protein, enzyme activity assay by spectrophotometric methods. SDS-PAGE, western blotting, protein quantitation and discussion on characterisation techniques by spectrophotometric advanced methods.

Molecular Biology Module

The Molecular Biology Module will focus on second and third generation DNA sequencing technologies.
The module focuses on experimental and computational aspects of DNA/RNA sequencing.
Introduction to sequencing technologies:

• Illumina (HISeq, MiSeq), Roche (454), Life Technologies (Ion Torrent).

Sample preparation:

• miRNA-seq
• mRNA seq
• whole transcriptome sequencing
• directional sequencing
• enrichment protocols
• DNA exome
• DNA whole genome

Bioinformatics analysis approaches

• Mapping
• Filtering
• Differential expression
• SNP calls

Examples of the above mentioned technologies and analytical methods
Biochemistry method: theoretical lessons 8 hours. Protein purification laboratory 16 hours.
Molecular Biology Module: theoretical lessons 8 hours, laboratory and data analysis 16 hours

Biochemistry

Rob Reed, David Holmes, Jonathan Weyers, Allan Jones Metodologie di base per le scienze biomolecolari, Zanichelli (2002).

oppure

A.J. Ninfa, D.P. Ballou. Metodologie di base per la biochimica e la biotecnologia. Zanichelli (2000).

oppure

IC Wilson, J. Walker. Metodologia biochimica: le tecniche biochimiche in laboratorio. Ed. Raffaello Cortina (1995).

Molecular Biology

Material provided during lessons.