Degree course: 
Corso di First cycle degree in BIOLOGICAL SCIENCES
Academic year when starting the degree: 
Academic year in which the course will be held: 
Course type: 
Basic compulsory subjects
First Semester
Standard lectures hours: 
Detail of lecture’s hours: 
Lesson (72 hours)

It is recommended to begin the course with a strong background in Cytology and Histology, Organic Chemistry and Genetics.

Voto Finale

The Course of Recombinant DNA technology is aimed at providing the students with the theoretical bases of recombinant DNA technology, coupled to a detailed knowledge of the underlying experimental approaches.
The expected learning outcomes for this course will be the following:
- Knowledge of the theoretical/operative know-how on the main methodologies for DNA manipulation-
- Ability to properly develop an experimental plan within the context of the current research in the fields of molecular genetics and molecular biology.
- Ability to achieve informed judgment, adequate expertise and communication skills in relation to the course content, and the skills required to develop and maintain issues related to the acquired knowledge, by means of critical reasoning and problem-solving attitudes.
- Ability to implement basic experimental protocols during laboratory classes and at the same time to develop a team-work attitude.

• DNA as an informational molecule (2 hours)
• Molecular cloning: history and key concepts (2 hours)
• From natural plasmids to cloning vectors (1 hour)
• Restriction enzymes (1 hour)
• The four cloning steps (2 hours)
• Preparation and analysis of recombinant DNA (0,5 hours)
• Second and third generation plasmid-based cloning vectors. (2 hours)
• High-capacity cloning vectors: lambda, P1 and cosmid vectors. PAC, YACs and BACs). Viral vectors for eucaryotic cells (2 hours)
• Principles of molecular hybridization assays (1 hour)
• Applications hybridization -1: Southern, northern and zoo blot. Colony hybridization (2 hours)
• Applications hybridization -2: FISH assays, RNA ISH, comparative genomic hybridization (CGH), microarray hybridization (2 hours)
• Applications of PCR: Mutation screening and detection, genomic/cDNA screening, RT-PCR, cloning by PCR, DOP-PCR, in situ PCR. Outline on digital PCR (3 hours)
• Real-time PCR and digital : principles and applications (2 hours)
• Genomic DNA libraries: introduction and mode of assembly. Library complexity. cDNA libraries (1,5 hours)
• Methods for DNA mutagenesis (2 hours)
• Gene transfer assays in eucaryotic cells: calcium phosphate transfection, lipofection, electro-poration and viral vectors infections(1 hour)
• Methods for the identification of a gene’s regulatory elements: DNase hypersensitive site’s mapping, gene transfer with reporter vectors, DNA footprinting, Electrophoretic Mobility Shift Assay (EMSA) (3 hours)
• Introduction to the key model organisms in experimental biology. Key concepts for transgenesis. Methods for producing and analyzing transgenic mice (2 hours)
• Transgenic systems with inducible gene expression(2 hours)
• Gene targeting principles and approaches. Gene “knock-out” e “knock-in” in mouse model systems. Conditional gene knock-out (3 hours)
• Use of chromosome engineering and zinc-finger nucleases, TALEN and CRISPR assays for gene targeting in vivo (1 hour)
• Genetic maps and molecular markers. (1 hour)
• DNA profiling (1 hours)

Laboratory lessons (1 CFU, 16 hours)
Four practical laboratory training lessons (4 hours each) will be held. The teaching module is focused on the planning and fulfillment of a molecular cloning experiment, under the following schedule:
• Day 1: Planning of the experimental strategy. Digestion of DNA templates with restriction enzymes, dephosphorilation of the plasmid vector, preparative gel electrophoresis and DNA gel extraction
• Day 2: quantitation of the extracted DNA samples. Planning and execution of the ligation reaction. Bacterial transformation.
• Day 3: analysis of the results. Colony PCR to discriminate recombinant and nonrecombinant clones. Inoculation of PCR-positive clones in liquid medium.
• Day 4: Mini-prep of plasmid DNA. Electrophoretic analysis of the recombinant DNA molecole. Final discussion of the whole data.

Recommended textbook: Watson/Caudy/Myers/Witkowski “DNA ricombinante” – Zanichelli. Other textbooks might be suggested for in-depth study. The teaching material is updated regularly and will be provided to all students in the e-learning online platform as Powerpoint slides file, short notes, animation files and articles from scientific literature on selected issues. “Brain training” questions will also be uploaded in the online platform.