The course starts with the basic principles of recombinant DNA illustrating the mechanism of action of of restriction and modification enzymes, and gene cloning through host-vector systems.
The mechanism of action of standard and quantitative PCR is presented in detail, but particular importance is given to applications of such methodologies, through specific examples.
Among the different methods of DNA sequencing only the Sanger method is reported, both in its manual and automated versions.
The various techniques of analysis of nucleic acids and proteins, including electrophoresis on agarose and polyacrylamide and through blotting (Southern, Northern and Western blotting), and the different fields of application of these techniques are presented.
In regard to libreries, both the construction process, and the analysis of the genomic, cDNA and expression libraries are shown, with an emphasis on information obtained from their use.
The Arabidopsis thaliana and Homo sapiens genome projects are presented, explaining the criteria of choice of model organisms, and information obtained by the complete knowledge of these genomes.
Structural, functional and comparative genomics, as well as the technologies related to each of these branches, are presented. In particular, technologies related to structural genomics, through analysis of the genomic libraries for the sequencing of extended portions of DNA, and to functional genomics, through the main techniques of analysis of transcriptome and proteome, are explained.
Finally, genetically modified plants are presented: how they are realized through the two main systems (biolistic and Agrobacterium), by illustrating advantages and disadvantages of both these methods; how a genetic construct for expression in plants is made, which genetically modified plants are present on the market, with hints related to the issue of their acceptability.

Practical classes:
• Bacterial transformation using a recombinant plasmid vector, which is extracted in a subsequent practical class and characterized by agarose gel electrophoresis
• Standard PCR to determine the presence of a specific transgene in plants of GM wheats
• SDS-PAGE to analyze the protein composition of wheat kernels
• The use of the spectrophotometer to determine the amount of DNA and proteins

If the students are more than 25, practical classes are performed in turns.

Brown T. Molecular Biotechnology
Slides and other materials will be made available to students on Moodle platform

Biotechnology for the improvement of crop production.
Introduction to the role of biotechnologies applied to plant production with reference to genetic engineering, in vitro cell and tissue cultures, to technologies for diagnosis and traceability, to the study of genetic variability for phylogenesis, varietal characterization and management of germplasm, to the techniques for manipulating protoplasts and obtaining interspecific hybrids.
Molecular markers and genetic analysis.
Concept of genetic marker and introduction to molecular polymorphism analysis.
Biochemical markers. Molecular markers, hybridization and PCR, RFLP and VNTR markers, RAPD, SCAR, CAPS, AFLP, SSR, ISSR, SNP markers.
Use of molecular markers
Studies of formal genetics and development of association maps;
search for markers associated with loci that control qualitative and quantitative characters;
positional cloning of Mendelian and QTL genes;
marker assisted selection for genetic improvement;
linkage disequilibrium and mapping by association;
genetic variability analysis, genetic distances and clustering methods;
varietal characterization and gene flow analysis.
Practice topics.
In silico research of microsatellite markers and identification of primer pairs to amplify the SSR region.
Search for single nucleotide polymorphisms from EST libraries by sequence alignment.
Screening of an F2 population segregating morphological and molecular markers, phenotyping, genotyping (DNA extraction, PCR, electrophoresis), data analysis.

Barcaccia & Falcinelli - Genetica e genomica Vol. III - Liguori editore.
Material provided by the teacher during the course.