MOLECULAR BIOTECHNOLOGY
Academic Year 2022/2023 - Teacher: Angela Roberta LO PIEROExpected Learning Outcomes
The educational objective of the course is to provide students with theoretical and practical knowledge of the main techniques used to analyze the structure and function of biomolecules, in particular those that led to the development of new disciplines such as genomics, transcriptomics and proteomics.In particular, the expected learning outcomes declined according to the Dublin Descriptors are:
Knowledge and understanding
At the end of the course, the student will have acquired basic knowledge on the main experimental methodologies in the field of molecular biotechnologies applied to plants. He/she will acquire knowledge and understanding of the role of nucleic acids and proteins and their analysis in cellular processes under normal and stressful conditions.
Applying knowledge and understanding
At the end of the course, the student will be able to translate theoretical knowledge into methodological tools for solving scientific problems and will be able to process experimental data and represent them in the most suitable way. The ability to apply knowledge and understanding will be assessed through short reports to be carried out after the laboratory activities and by carrying out the two questions relating to experimental problems included in the final examination.
Making judgements
At the end of the course, the student will be able to answer questions, develop a reasoning, describe the results of an experimental test and start a process of data interpretation.
Communication skills
The student will be able to describe, discuss and define the applicability of theoretical topics included in the course contents. These skills will be assessed through the two open questions inserted in the final examination.
Learning skills
At the end of the course, the student will be able to develop a logical reasoning and measure himself/herself with new information and to use it for different purposes.
Course Structure
Teaching (8 CFU) includes 42 hours of frontal lessons and 28 hours of laboratory exercises. For lectures, the teacher will make use of power point presentations and videos, also in English. The laboratory exercises will take place in the form of a mini-course on the main topics of the course (for example: extraction of nucleic acids, electrophoresis of nucleic acids and proteins, preparation of plasmid DNA and digestion with restriction enzymes),
As a guarantee of equal opportunities and in compliance with current laws, interested students can ask for a personal interview in order to plan any compensatory and/or dispensatory measures, based on their specific needs and on teaching objectives of the discipline. It is also possible to ask the departmental contacts of CInAP (Center for Active and Participatory Inclusion - Services for Disabilities and/or DSAs).
Required Prerequisites
Attendance of Lessons
Attendance of the lessons is not compulsory but strongly recommended
Detailed Course Content
Basic concepts on the structure and functions of nucleic acids and proteins.The manipulation of nucleic acids. Southern and Northern blots. Molecular and genomic markers. Qualitative and quantitative PCR. Cloning techniques and vectors. The genetic transformation of prokaryotes. Preparation and analysis of cDNA and genomic libraries. The DNA microarray technology. Subtractive hybridization. Methods of nucleic acid sequencing. Genomics and Transcriptomics. RNAseq. Epigenomics, Protein two-dimensional electrophoresis and proteomics. The genetic transformation of eukaryotes. Cisgenesis and Intragenesis. Genome editing. Expression Vectors and fusion proteins. The recombinant DNA technology in the food industry.
Textbook Information
1) Biochimica e biologia molecolare: Principi e Tecniche, Wilson K., Walker J., ed. Raffaello Cortina (ottava edizione).
2) Rao R., Leone A., Biotecnologie e genomica delle piante, Idelson-Gnocchi ed.
3) Scientific articles provided by the teacher
Course Planning
| Subjects | Text References | |
|---|---|---|
| 1 | Generalità su struttura e funzione degli acidi nucleici, replicazione del DNA, le classi di RNA (coding e non-coding), la sintesi proteica. | Testo 1: cap 4 Testo 2: cap 1 |
| 2 | Le proprietà fisico-chimiche delle basi azotate dei nucleotidi: distribuzione elettronica ed assorbimento della luce; denaturazione e rinaturazione degli acidi nucleici; calcolo del Tm; influenza della composizione in basi e della forza ionica sul Tm | Testo 1: cap 4 |
| 3 | Gli enzimi impiegati nella tecnologia del DNA ricombinante: un’overview. Endonucleasi di restrizione, DNA ligasi, DNA polimerasi (I, II, III, IV e V), frammento di Klenow, trascrittasi inversa, fosfatasi alcalina, trasferasi terminale, metilasi | Testo 1: cap 4 |
| 4 | Isolamento e separazione degli acidi nucleici: Isolamento del DNA da organismi procarioti ed eucarioti. Preparazione di DNA plasmidico. Isolamento dell’RNA totale e purificazione dell’mRNA tramite cromatografia di affinità | Testo 1: cap 4 |
| 5 | Vettori per il clonaggio genico: plasmidi, fagi, cosmidi, vettori BAC e YAC La trasformazione batterica e la selezione dei batteri ricombinanti: selezione con doppio antibiotico ed alfa-complementazione | Testo 1: cap 4 |
| 6 | Elettroforesi degli acidi nucleici in gel di agarosio e di poliacrilamide | Testo 1: cap 6 |
| 7 | La polymerase chain reaction PCR: principio ed applicazioni. Design di primers, allestimento di una reazione di PCR; Analisi dei prodotti di PCR su gel di agarosio. La RT -PCR. La PCR quantitativa: real time PCR | Testo 1: cap 4 Testo 2: cap 2 |
| 8 | Southern e Northern blots, dot blots, trasferimento di DNA da placche di lisi fagica. Costruzione di librerie. Isolamento ed identificazione di geni clonati. Cenni sull’utilizzo dei tools bioinformatici di NCBI (ORF finder, BLAST) | Testo 1: cap 4 Testo 2: cap 2 |
| 9 | Design e marcatura di sonde geniche con metodologie alternative al 32P: sistema biotina-streptavidina, marcatura con digossigenina. Enzimi reporter: fosfatasi alcalina e perossidasi di rafano | Testo 1: cap 4-9 |
| 10 | I metodi di sequenziamento del DNA: il metodo dei dideossiribonucleotidi (Sanger), il pirosequenziamento. PCR in emulsione, Bridge PCR. Le piattaforme di Next Generation Sequencing | Testo 1: cap. -20 Testo 2: cap 2 |
| 11 | L’uso del microarray in studi di genomica funzionale e strutturale. L’analisi globale del trascrittoma: RNAseq. Metodi di arricchimento: la ibridizzazione soppressiva sottrativa (SSH) e la microdissezione laser. | Testo 1: cap. 4-20 Testo 2: cap 2 |
| 12 | La trasformazione genetica degli eucarioti. Cisgenesis ed Intragenesis. Genome editing: tecnologia CRISP/cas9. Cenni sui sistemi meganucleasi, nucleasi a dita di zinco e TALEN | Articoli scientifici forniti dal docente |
| 13 | La produzione di proteine da geni clonati: vettori di espressione. Produzione di proteine di fusione (GST-tag, His-tag). | Testo 1: cap. 5 Testo 2: cap 2 |
| 14 | Elettroforesi delle proteine in gel di poliacrilamide. La separazione delle proteine in base al loro punto isoelettrico: isoelectrofocusing (IEF). Elettroforesi delle proteine bidimensionale: analisi del proteoma, MALDI-TOF. Proteomica | Testo 1: cap. 21 Testo 2: cap 2 |
| 15 | Marcatori molecolari: definizione e classificazione. Cenni sui marcatori RFLP, SSR, RAPD, e SNP. | Testo 2: cap 2 |