Learning objectives
Learning about fundamental and advanced techniques used for the isolation and determination of biological macomolecules, in particular DNA and proteins.
Course unit content
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1. The biochemical laboratory: chemical risk and biological risk. Cell cultures. Sterilizing materials and ensuring sterile conditions. <br />
2. Recombinant DNA technology: host organisms, restriction endonucleases, DNA ligases, cloning vectors, plasmids as cloning vectors, recombinant selection, alpha-complementation, PCR, site-specific mutagenesis, DNA-microarrays, gene libraries. <br />
3. Recombinant protein expression: parameters affecting the quantity of protein expressed, expression vectors, cell strains, LAC and T7 promoters, expression induction, cell-free expression, fusion proteins. Recombinant proteins as drugs. <br />
4. Protein purification: <br />
* Extraction techniques: mechanical and non-mechanical methods. <br />
* Centrifugation techniques: principles, types of centrifuge, differential sedimentation, gradient centrifugation. <br />
* Solution concentration and pad change: ultrafiltration, dialysis, diafiltration. <br />
* Methods of fractioning: by stability and by solubility (salting out, by pI, and by the addition of organic solvents). <br />
* Chromatography: principles and techniques. Ion exchange chromatography, hydrophobic interaction chromatography, inverse phase chromatography, size exclusion chromatography and its applications, affinity chromatography (IMAC, protein A, GST) <br />
5. Electrophoretic techniques: principles and applications for biological macromolecules. Mobile phase and zone electrophoresis, solid-phase substrates, protein electrophoresis (in native conditions, and SDS-PAGE with applications), protein band detection, Western blotting, isoelectrofocalization, DNA electrophoresis, DNA band detection, an outline of restriction analysis and its applications, Northern blotting, Southern blotting, DNA sequencing. <br />
6. Two-dimensional electrophoresis and mass spectrometry. <br />
7. Spectroscopic techniques: <br />
* Absorption spectroscopy: the Lambert-Beer law, layout of a colorimeter and a spectrophotometer. Protein absorption spectra. Colorimetric assays for protein concentration determination. <br />
* Fluorescence spectroscopy: principles (emission from singlet and triplet state, Stokes shift, quantum fluorescence yield), applications (study of proteic dynamics, collisional quenching, and exposure of fluorophores to solvent, FRET and calcium sensors). <br />
* Circular dichroism: principles (linear radiation polarization, circular radiation polarization, elliptical radiation polarization and ellipticity). Applications in the study of protein structure. <br />
* NMR: an outline of its applications in the study of protein dynamics and structure. Isotopic protein enrichment for expression in minimal media. <br />
8. Enzymatic techniques: <br />
* Studies on stationary state: continuous and discontinuous methods. Direct, indirect and coupled assay. Chromogenic substrates. Radioisotopic methods and product separation. <br />
* Studies on pre-stationary state: rapid mixing methods (stopped-flow) and relaxation methods (pH-jump with caged-H+ compounds). <br />
9. Chemical immune techniques: antibodies (structure, specificity, marking). The production of monoclonal and polyclonal antibodies. Immune dosage levels: RIA and ELISA. Applications: pregnancy test and HIV test. <br />
10. Radioactive isotopes: their use, and measuring radioactivity. <br />
11. Protein crystallization: principles and methods. X-ray diffusion, diffraction maps, electron density maps. Protein data bank: an outline of the use and display of three-dimensional protein structure. <br />
Bibliography
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Wilson and Walker: “Biochimica e Biologia Molecolare", new Italian edition. Cortina Editore. <br />
Reed et al.: “Metodologie di base per le scienze biomolecolari”. Zanichelli <br />
T.A. Brown. “Biotecnologie Molecolari: Principi e Tecniche”. Zanichelli <br />
NELSON e COX: “I principi di biochimica di Lehninger”, fourth edition. Chapter 9. Zanichelli <br />
