Biochemical and biomolecular techniques

Students will acquire both theoretical/practical knowledge of basic biochemical and biomolecular methodologies useful for experimental analysis and application skills used in the field of biomolecular disciplines. During the scheduled laboratory hours, students will acquire practical skills in the use of the biomolecular techniques, with particular attention to the applications of genetic engineering and recombinant proteins.

Frontal lessons and laboratory. Wheter lessons are given in a mixed or remote mode, necessary changes may be introduced in order to respect the program reported in the Syllabus.

It is preferable to have acquired the main topics of Biochemistry and Molecular Biology.

• Manipulation of nucleic acids: extraction and isolation of DNA and RNA from biological samples; use of enzymes (nuclease, ligase, polymerase, restriction endonuclease).
• Principles of gene cloning: plasmid and phage-based vectors; selection of recombinants methods; model organisms; transformation of bacteria; cell transfection.
• PCR and DNA visualization: PCR; use of PCR in gene cloning; PCR mutagenesis; quantitative PCR; reverse-transcription PCR and cDNA synthesis; agarose gel electrophoresis.
• Hybridization techniques: marking of the probes and detection; screening of genetic libraries; southern and norten blot; microarray.
• DNA sequencing: Sanger sequencing; next-generation sequencing; sequencing of whole genomes (shotgun approach and hierarchical shotgun).
• Proteome analysis: protein extraction; SDS-PAGE; 2D-PAGE; BN-PAGE; proteomic approach.
• Immunological techniques: primary and secondary antibodies; types of conjugation; western blot; general principles of ELISA and immunofluorescence.
• Production of recombinant proteins: expression vectors; heterologous expression in bacteria; fusion proteins; purification of proteins by affinity chromatography; use of eukaryotic cells for production of recombinant proteins (yeast, insect cells).

Biotecnologie molecolari. Principi e tecniche. Brown,  Zanichelli editore.

DNA ricombinante. Watson, Caudy, Myers & Witkowski, Zanichelli editore.

Genomi 3. Brown, EdiSes editore.