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: 
Standard lectures hours: 

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

Final Examination: 

At the end of the module, the student will undergo an oral examination. During the exam, the acquired knowledge will be evaluated by raising at least three question focussed on different issues. Questions aimed at evaluating the problem-solving ability of the student will also be posed. Solution of a “brain-training” question previously posted by the teacher in the dedicated e-learning web system might also be required to the student. A specific question over the laboratory module will also be asked. For each student, the final judgement will consider the quality and precision of the answers (70%), the ability to motivate statements (20%) and teh communication skills (10%). The time for the exam is about 20-25 minutes and the exam will be considered passed equal or over the 18/30 mark.

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)

Four practical laboratory training lessons (4 hours each) will be held. The teaching module is focused on the planning and fulfillment of an experimental plan that wil include the following experiences:
- PCR-mediated analysis of the results of a molecular cloning experiment
- DNA extraction from bacterial clones and analysis by restriction enzymes digestions
- Gene transfer experiments in human cultured cells by means of transient transfection assays
- Validation of the transfection efficacy by realtime PCR gene expression analysis
- Further validation of the transgfection efficacy by fluorescence microscopy analysis

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.

The Recombinant DNA technologt module will be split in normal classes (5 CFU) and an experimental laboratory module (1 CFU). Class lessons will be held with the aid of slide presentation sessions, coupled to projection of didactic movies when required.
The experimental laboratory module will be held in the Experimental Biology Lab at the Department of Biotechnology and Life Sciences, via JH Dunant 3, Varese. Each student will be assigned a workstation endowed will all necessary equipment for the implementation of the proposed experimental plan. A printed tutorial guide will also be distributed to the students. Lab attendance is mandatory for all students, which can skip no more than one lesson for explained reasons. Students are required to attend the laboratory lessons with a personal lab coat. Students with known intolerance or allergy to drugs or chemicals that might be stored in a research lab must absolutely inform the teacher before the beginning of the lab module.

The teacher will answer questions regarding the topics discussed in the course following an arrangement either by phone or e-mail. Students are kindly required not to ask bureaucratic/administrative question, if not really urgent. Email address: