Degree course: 
Corso di First cycle degree in BIOTECHNOLOGY
Academic year when starting the degree: 
Academic year in which the course will be held: 
Course type: 
Supplementary compulsory subjects
Second semester
Standard lectures hours: 
Detail of lecture’s hours: 
Lesson (40 hours), Laboratory (16 hours)

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

At completion 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 focused 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 will 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 the communication skills (10%). The predicted time for each exam is about 20-25 minutes and the exam will be considered passed if the student will attain a final mark equal or over the 18/30 threshold.

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 bases of the main methodologies for DNA manipulation (Recombinant DNA Technologies class).
- Ability to properly develop an experimental plan within the context of the current research in the fields of molecular genetics and molecular biology (Recombinant DNA Technologies class).
- Ability to achieve informed judgment, adequate expertise and communication skills in relation to the course content for both modules, 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: summary (2 hours)
• DNA ligase-independent cloning strategies(1 hour)
• Second and third generation plasmid-based cloning vectors. Expression vectors (2 hours)
• High-capacity cloning tools: lambda, P1 and cosmid vectors. PAC, Yeast Artificial Chromosomes (YACs) and Bacterial Artificial Chromosomes (BACs). Viral vectors for eucaryotic cells (2 hours)
• Principles of molecular hybridization assays (1 hour)
• Applications of molecular hybridization techniques – 1: Southern, northern and zoo blot. Colony hybridization (2 hours)
• Applications of molecular hybridization techniques – 2: fluorescence labelling of nucleic acids and fluorescence in situ hybridization (FISH), RNA in situ hybridization (RNA ISH), comparative genomic hybridization (CGH), microarray hybridization (2 hours)
• Introduction and applications of PCR: Mutation screening and detection, genomic/cDNA screening, reverse transcription PCR, cloning by PCR, DOP-PCR. (2 hours)
• Real-time and digital PCR : principles and experimental approaches (2 hours)
• Whole genome amplification by PCR: DOP-WGA and Multiple Displacement Amplification (MDA) (2 hours)
• Genomic DNA libraries: introduction and mode of assembly. Library complexity and genome equivalent values. cDNA libraries (2 hours)
• Methods for DNA mutagenesis: (2 hours)
• Gene transfer assays in eucaryotic cells (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), Chromatin conformation Capture (CCC) (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)
• Chromosome engineering. Genome editing by means of and zinc-finger nucleases, TALEN and CRISPR/Cas9 assays(2 hours)
• Genetic maps and molecular markers. (2 hours)
• DNA profiling (1 hours)

OUTLINE OF THE EXPERIMENT: subcloning of a specific protein coding sequence into a mammalian expression vector, to express it as a fusion with GFP, transfection of the construct in a human cell line and analysis of its expression by fluorescence microscopy and by qPCR.

• analysis of restriction sites using specific programs;
• digestion with restriction endonucleases;
• agarose gel electrophoresis;
• purification of DNA fragments;
• ligation
• Transformation of ligated products into E. coli;
• solid and liquid cultures of bacteria
• Plasmid DNA extraction and purification
• Transfection of the prepared constructs into a human cell line
• Observation of transfected cells at an epifluorescence inverted microscope
• qPCR for relative quantification of the target transcript of interest in cells and calculation of its fold increase using the 2ˆ-DeltaDelta Cq method (Livak 2001).

Recommended textbook:
1) Watson/Caudy/Myers/Witkowski “DNA ricombinante” – Zanichelli.
2) Maccarone M. “Metodologie biochimiche e biomolecolari” Zanichelli
3) Strachan-Read “Genetica molecolare umana” Zanichelli

Other textbooks might be suggested for in-depth study. The teaching material is updated regularly and will be provided to all students as Powerpoint files, short notes, animation files and articles from scientific literature on selected issues. The abovementioned material will be made available to all students via the e-learning online platform. “Brain training” questions will be uploaded in the online platform. Printed folders describing each exercise to be carried out in the laboratory module will also be provided to each student.


The module will be split in standard class lessons (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, who can skip no more than one lesson. 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 present in a research lab must absolutely inform the teacher before the beginning of the lab module.

Both teachers will answer questions regarding the topics discussed in the course following an appointment by e-mail. (;