TECNICHE BIOCHIMICHE E BIOMOLECOLARI

The student will acquire skills related to the techniques for the analysis of the biochemical aspects of prokaryotes and eukaryotes and cell culture techniques, even on a large scale; for the study of the structure, functions and analysis of biological macromolecules and cellular processes.

Cell cultures: primary cell cultures and cell lines.

Cell cycle analysis, cell proliferation, vitality and cell death assays.

Chain Polymerization Reaction (PCR), cDNA synthesis by reverse transcription. Bioinformatics parameters and softwares for PCR Primer Design (Primer 3 NCBI).

Qualitative and quantitative analysis of transcripts.

Evaluation of polymorphisms by quantitative PCR or "real-time PCR"

Digestion reactions by restriction enzymes. Reactions by modification enzymes.

Extraction, Purification and qualitative and quantitative determination of nucleic acids and proteins

Electrophoresis of nucleic acids and proteins.

Hybridization techniques. Southern blot, Northern blot and Western blot.

DNA or RNA sample preparations for global genome analysis and microarrays

Nucleic Acids Sequencing: Sanger and Next Generation Sequencing

Analysis of epigenetic modification (methylation)

Transient and stable transfection techniques and silencing of gene expression by "RNA interference"

Recombinant DNA technology and cloning: Preparation of a recombinant plasmid vector; Bacterial transformation and extraction of plasmid DNA

Enzymatic activity tests in biological samples.

Databases.

Biochemical and biomolecular laboratory techniques in medical molecular diagnostics.

Laboratory Experiences

Esp 1 human Cell lines

Esp 2 Nucleic Acids quantitative assay

Esp 4 Electrophoresis in agarose gel

Esp 3 DNA extraction from human cell cultures

Esp 5 cDNA synthesis

Esp 9 Transfection and Silencing

Esp 7 Digestion with Restriction Enzymes

Esp 6 Polymerase Chain Reaction (PCR)

Exp 8 Quantitative PCR / qRT Allelic Discrimination

Fundamental Molecular Biology Lizabeth A. Allison

Additional material supplied by the teacher

Biologia molecolare Autori: Francesco Amaldi, Piero Benedetti, Graziano Pesole, Paolo Plevani, II eds Casa Editrice Ambrosiana (2014) – Zanichelli

Metodologie biochimiche. Principi e tecniche per l'espressione, la purificazione e la caratterizzazione delle proteine Autori : Martino L. Di Salvo, M. Carmela Bonaccorsi di Patti, Roberto Contestabile. Editore: CEA