APPLIED BIOCHEMISTRY
Academic year and teacher
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- Versione italiana
- Academic year
- 2022/2023
- Teacher
- ALESSIA FINOTTI
- Credits
- 9
- Didactic period
- Secondo Semestre
- SSD
- BIO/10
Training objectives
- Knowledges: most used molecular biology technology employed in genetic engineering and for the production of recombinant DNA. The second training objective is to describe biochemical and molecular biology technologies for the study of gene expression. A third objective is to describe the mechanism of action of biotechnological drugs based on DNA molecules.
Skills: ability to perform basic experiments of applied cell biology, biochemistry and molecular biology of nucleic acids and macromolecules of biotechnological interest. Ability to update the knowledge on the development of new therapeutic approaches.
METHODOLOGIES FOR THE TEACHING OF BIOCHEMISTRY AND APPLIED BIOCHEMISTRY
A. Critical discussion of the most important methods for the construction of teaching in the biochemistry field, based on experimental approaches and observation of natural phenomena.
B. Description of the most important discoveries in the field of biochemistry, applied genetics, genetic engineering, molecular biology.
C. Methods and technologies for the study of biochemistry and applied biochemistry with respect to society, environment, health, biotechnology.
D. Teaching and learning processes in respect to digital technologies, e-learning and multimedia platforms.
D. I livelli di disseminazione e divulgazione degli argomenti attinenti alla biochimica applicata.
E. Teaching methodologies for the improvement of specific definitions in the field. Prerequisites
- The student should be expert in the following fields: structure and functions of DNA, RNA and protein; RNA; DNA replication; transcription and splicing; protein synthesis.
Course programme
- BIOCHEMICAL TECHNOLOGIES FOR STUDIES ON GENE PRODUCTS
-DNA extraction and purification
-DNA quantification and analysis
-mRNA and other cellular RNAs extraction and purification
-RNA quantification and analysis
-Protein extraction and quantification
-Enzyme for nucleic acids manipulation (applications)
BIOMOLECULAR TECHNOLOGIES FOR STUDIES ON GENOME STRUCTURE AND GENES EXPRESSION AND FUNCTIONS (Part 1)
-PCR
-RT-PCR
-Quantitative Real-Time PCR (qPCR)
-Digital PCR
-DNA sequencing
BIOMOLECULAR TECHNOLOGIES FOR STUDIES ON GENOME STRUCTURE AND GENES EXPRESSION AND FUNCTIONS (Part 2)
-Southern Blotting
-Northern Blotting
-Western Blotting
BIOMOLECULAR TECHNOLOGIES FOR STUDIES ON GENOME STRUCTURE AND GENES EXPRESSION AND FUNCTIONS (Part 3)
-Other techniques for transcripts analysis
-Essays for gene expression regulation studies
-Essays for studies of gene functions
RECOMBINANT DNA AND GENETIC ENGINEERING
-Basics of DNA cloning
-Cloning vectors
-Expression vectors and recombinant proteins
-OGM and transgenic animals
-Therapeutic recombinant protein (examples)
GENE THERAPY: BASIC CONCEPTS
-Introduction to the gene therapy concept
-Gene transfer methods
-Stem cells for gene therapy and regenerative medicine
GENE THERAPY: SYNTHETIC OLIGONUCLEOTIDES APPLICATIONS
- Antigene strategy with Triplex Forming Oligonucleotides (TFO)
- Antisense strategy with oligonucleotides (ASO)
¬ -Antisense strategy (RNA interference, siRNA and miRNA)
- The decoy strategy
- Aptamers
GENE THERAPY: GENOME EDITING
-Basics and aims
-CRISPR/Cas9
-Examples of GE for therapy of human diseases
MOLECULAR DIAGNOSIS AND EXPERIMENTAL THERAPY
- Neoplastic diseases
- Thalassemia
Practical training in laboratory (12 hours):
The hours of practical training in the laboratory will give the opportunity to the students to realize the theoretical knowledge acquired and will be based on some of the methods (RNA extraction, retro-transcription of RNA into cDNA, quantitative PCR) most used in research laboratories and simultaneously learn how to use scientific instrumentations related to these methodologies. In particular, students will learn to use the pipettes (p1000, p200, p20) and they take on the experimental steps to analyze the expression of a specific gene target: from the RNA extraction, its quantification, both using the spectrophotometer and DeNovix instrument, until the qualitative analysis of RNA by electrophoresis on agarose gel; finally, the production of cDNA by reverse transcription and PCR quantification of a specific mRNA. Didactic methods
- The course is organized with lectures (64 hours) on the program topics and guided laboratory exercises (12 hours). The course objective is not only to teach the student the cultural bases of the course but also teach the "know-how" through laboratory classes and lectures that, also using video as a teaching material, allow you to have the visual perception of the experiments that they are performed in biochemical studies and molecular biology applied to the development of new drugs and therapeutic approaches.
Laboratory:
The students are divided (about 24 students per turn). The students will have the opportunity to work alone or in pairs about the following methods: RNA extraction, retro-transcription reaction, PCR reactions and data analysis. quantification, analysis on agarose gel. Learning assessment procedures
- Oral examination. The aim of the oral examination is to test the level of knowledge and deepening of the topics of the course program and the reasoning skills developed by the student on issues related to molecular diagnosis based on applied biochemistry and molecular biology. Generally, three arguments are asked, to which, in order to obtain the minimum grade, it is necessary to exhaustively respond. For each question 10/30 are attributed. The oral examination may finish also with one only question if the commission verifies serious deficiencies of basic topics of the course. The overall evaluation is expressed in thirtieths (minimum grade 18, maximum grade 30 cum Laude).
Reference texts
- 1) Files of the lessons (pdf format) will be provided in the Google Classroom of the course.
2) Scientific articles, websites, links or useful references will be indicated and uploaded in the Classroom.
3) Textbooks:
-Tecniche e metodi per la biologia molecolare. Amaldi, Benedetti, Pesole, Plevani. 2020. Casa Editrice Ambrosiana-ZANICHELLI
-Metodologie biochimiche e biomolecolari. M. Maccarrone-ZANICHELLI
-Metodologie biochimiche. Espressione, purificazione e caratterizzazione delle proteine. Bonaccorsi di Patti, Contestabile, Di Salvo-ZANICHELLI
-Metodologie biochimiche. Bonaccorsi di Patti, Contestabile, Di Salvo-Casa Editrice Ambrosiana
-Biotecnologie Molecolari. Principi e tecniche. Terry A. Brown–ZANICHELLI
-Farmaci Biotecnologici. Vegeto-Maggi-Minghetti. Casa Editrice Ambrosiana. Distribuzione esclusiva Zanichelli, 2020.
-Genomi 4.Terry A. Brown-IVedizione, 2018.EDISES
-DNA ricombinante. Geni e Genomi. J.D. Watson et al.-ZANICHELLI
-Analisi dei Geni e Genomi. Richerd J.Reece-EdiSES
-Compendio di Biotecnologie Farmaceutiche. Maria Luisa Calabrò-EdiSES
