Biochemical and biomolecular techniques

1. Knowledge and Understanding

By the end of the course, students will have acquired theoretical and practical knowledge of the principles underlying the main methodologies used in biochemical and biomolecular laboratories. In particular, they will be familiar with methods for the extraction, manipulation, amplification, and analysis of nucleic acids, the principles of gene cloning and hybridization techniques, as well as the main methods for protein extraction, separation, and analysis. Students will also understand the principles of immunological techniques applied to protein analysis and the strategies used for the expression and purification of recombinant proteins in prokaryotic and eukaryotic systems. Through laboratory activities, students will also gain knowledge of the principles and practical steps of selected basic experimental techniques, including bacterial transformation, plasmid DNA extraction, centrifugation, chromatography, and electrophoresis.

2. Applying Knowledge and Understanding

Students will be able to apply the knowledge acquired during the course to the performance and interpretation of basic experimental procedures in biochemistry and molecular biology. Through laboratory activities, they will acquire practical skills in bacterial transformation, plasmid DNA extraction and purification, separation by centrifugation and chromatographic techniques, and electrophoretic analysis of nucleic acids and proteins by agarose gel electrophoresis and SDS-PAGE. Students will also be able to understand and follow experimental protocols, correctly use basic laboratory equipment, and interpret experimental results. In addition, they will understand the main steps involved in more complex experimental strategies, such as gene cloning and recombinant protein expression and purification.

3. Making Judgements

Students will be able to critically evaluate results obtained using the main biochemical and biomolecular techniques, identifying relevant experimental variables, potential sources of error, and the importance of appropriate experimental controls. They will also be able to compare different methodologies and identify, among those covered during the course, the most appropriate experimental approach to address a specific biological question.

4. Communication Skills

Students will be able to clearly and accurately describe the principles, procedures, and applications of the main biochemical and biomolecular techniques, using appropriate scientific terminology. They will also be able to present and discuss experimental results, explaining the rationale of the approach used, the main steps of the experimental procedure, and the conclusions that can be drawn from the data obtained.

5. Learning Skills

Students will develop the skills required to independently expand their knowledge of biochemical and biomolecular methodologies, understand and use experimental protocols, and consult laboratory manuals, scientific literature, and specialized resources. They will also acquire the theoretical and practical foundations needed to approach more advanced experimental methodologies and to pursue further training in biochemistry, molecular biology, biotechnology, and related disciplines.

The course consists of lectures and practical activities carried out both in the classroom and in the laboratory. Should the course be delivered in a blended or online format, the necessary adjustments to the teaching methods described above may be introduced to ensure completion of the course content outlined in the Syllabus.

Students are recommended to have successfully completed the examinations in Biochemistry, Genetics, and Molecular Biology.

Course attendance is mandatory to the extent required by the Degree Programme Regulations.

• Nucleic acid manipulation: extraction and isolation of DNA and RNA from biological samples; use of laboratory enzymes (nucleases, ligases, polymerases, restriction endonucleases).
• Principles of gene cloning: plasmid and phage vectors; selection of recombinants; use of model organisms; transformation of bacterial cells; transfection of eukaryotic cells.
• PCR and DNA visualization: PCR; use of PCR in gene cloning; PCR-based mutagenesis techniques; quantitative PCR; reverse-transcription PCR and cDNA synthesis; agarose gel electrophoresis.
• Hybridization techniques: DNA probes and target DNA; probe labeling and detection of hybridization; screening of genomic libraries; Southern and Northern blotting; microarrays.
• Proteome analysis: protein extraction; SDS-PAGE; 2D-PAGE; BN-PAGE; proteomic approaches.
• Immunological techniques: primary and secondary antibodies; types of antibody conjugation; Western blotting; general principles of ELISA and immunofluorescence.
• Recombinant protein production: expression vectors; heterologous expression in E. coli; fusion proteins; protein purification by affinity chromatography; use of eukaryotic cells for recombinant protein production (yeast and insect cells).

Assessment consists of an oral examination. Should circumstances require it, the examination may also be conducted remotely.

• What is a restriction endonuclease and how is it used in gene cloning?
• What are the main features of a plasmid vector?
• What is the difference between the selection of transformants and the selection of recombinant clones?
• Describe the general principle of PCR and explain how it can be used in gene cloning.
• What is meant by nucleic acid hybridization and what are the principles underlying this process?
• What is a protein tag and how can it be used experimentally?
• How can antibodies be used for protein analysis?
• What is meant by heterologous protein expression?