APPLIED BIOCHEMISTRY
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- Versione italiana
- Academic year
- 2017/2018
- Teacher
- ROBERTO GAMBARI
- 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 proteins, DNA, RNA; DNA replication; transcription and splicing; protein synthesis.
Course programme
- 1. BIOCHEMICAL TECHNOLOGIES FOR STUDIES ON REGULATION OF GENE EXPRESSION (12 hours)
A. Isolation of DNA and analysis of nucleic acids; electrophoretic techniques
B. Isolation of mRNA
C. Acellular system for protein synthesis
D. Production of radioactive-labeling molecular probes
2. RECOMBINANT DNA AND GENETIC ENGINEERING (12 hours)
A. Restriction
B. Restriction enzymes
C. Intramolecular and intermolecular association
D. DNA recombinant vectors
E. Production of cDNA and genomic libraries
F. Screening
G. DNA sequencing
3. BIOMOLECULAR TECHNOLOGIES FOR STUDIES ON GENOME STRUCTURE AND GENOMIC EXPRESSION (10 hours)
A. Southern blotting
B. Northern blotting
C. Molecular basis of PCR (Polymerase-chain reaction)
D. TaqI polymerase, analysis of PCR products
E. Molecular diagnosis by PCR technology
F. RT-PCR
4. BIOMOLECULAR TECHNOLOGIES FOR STUDIES ON TRANSCRIPTIONAL REGULATION OF GENE EXPRESSION (8 hours)
A. DNA-PROTEIN INTERACTIONS (EMSA)
B. DNasi-footprinting
C. CAT assay
5. SYNTHETIC OLIGONUCLEOTIDES (APPLICATIONS) (6 hours)
A. Antisense RNA
B. Antisense oligodeoxynucleotides
C. DNA decoy and dumbell oligonucleotides
D. Triplex Forming Oligonucleotides (TFO)
E. Antitumor and antiviral therapy with synthetic oligonucleotides
F. Gene therapy
6. MOLECULAR DIAGNOSIS AND EXPERIMENTAL THERAPY: NEOPLASTIC DESEASES AND VIRAL INFECTIONS (6 hours)
A. Cancer: neoplastic transformation.
B. Viral and cellular oncogenes
C. Proto-oncogenes
D. Isolation and cloning of transforming sequences: the Weinberg technique.
E. The neoplastic cell
G. Biochemical basis of metastasis
7. MOLECULAR DIAGNOSIS AND EXPERIMENTAL THERAPY: THALASSEMIA (6 hours)
A. Human hemoglobins
B. Molecular basis of thalassemia
C. Beta and alfa-thalassemia
D. The Kan experiment
8. TRANSGENIC ANIMALS (4 hours)
A. Techniques for production of transgenic mice
B. Transgenic animals for drug production
C. Transgenic animals as experimental models of pathologies: onco-mouse
D. Transgenic animals for the thalassemia’s study
9. EXPRESSION OF DNA MOLECULES CLONED IN BACTERIA (4 hours)
A. Molecular parameters that condition the expression of DNA sequences cloned in bacteria
B. Promoter and RBS (ribosome binding site). Enzyme Bal31
C. ATG vectors
10. EMBRYONIC STEM CELLS IN REGENERATIVE THERAPY (4 hours)
A. Stem cells; isolation of stem cells; use of stem cells
B. Embryonic Stem cells
C. Mammalian cloning
Pratical 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 quantifcation, 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 quantification of a specific mRNA by real time quantitative PCR. 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), they will work individually, while the data analysis can be done using pairwise comparisons. Laboratory technician training lasts 4 hours for 3 days. The students will have the opportunity to work alone or in pairs about the following methods: RNA extraction, quantification, analysis on agarose gel, retro-transcription reaction, real time quantitative PCR reactions and data analysis.
For the FIT project:
The critical discussion of the most important methods for the construction of teaching in the biochemistry field will be based on experimental approaches and observation of natural phenomena. This parte of the course will be performed with multimedia platforms. 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).
Reference texts
- Watson J, Gilman M, Witkowski J e Zoller M., DNA RICOMBINANTE, Zanichelli
Gibson G. e Muse SV, INTRODUZIONE ALLA GENOMICA, Zanichelli
Cooper GM e Hausman RE, LA CELLULA, BIOLOGIA MOLECOLARE, PICCIN
