INTEGRATED COURSE IN BIOCHEMICAL AND RECOMBINANT DNA TECHNOLOGIES
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- Versione italiana
- Academic year
- 2019/2020
- Teacher
- ROBERTO GAMBARI
- Credits
- 12
- Curriculum
- BIOTECNOLOGIE PER LA SALUTE
- Didactic period
- Secondo Semestre
Training objectives
- Course of Molecular and Recombinant Technologies (Dr. Alessia Finotti):
The course is aimed at teaching the techniques and the basic methodologies to study and analyze nucleic acids and gene expression, with special attention to the recombinant DNA technology.
The practical laboratory exercise will give the possibility to the student to apply the acquired knowledge and will focus on the most used methods in the common research laboratories. At the same time students will learn the use of basic instruments (e.g. the use pipettes and other common laboratory instruments), and will be addressed on the experimental steps for nucleic acids extraction (in particular RNA from biological matrices) and for their quali-quantitative analysis.
KNOWLEDGE AND UNDERSTANDING
The student will learn:
- correct use of scientific terms
- the techniques used to purify and quantify nucleic acids.
- the techniques used for genomic and transcriptomic analyses
- the basic elements to build a recombinant vector
- the different type of recombinant vectors.
ABILITY TO APPLY KNOWLEDGE AND UNDERSTANDING
The student will know:
- the DNA and RNA extraction methods and for its analysis
- the approach for genomics and transcriptomics analysis
- which technique should be used for gene cloning experiments
- the suitable vectors used for recombinant protein production
- the vectors used to analyze and modify gene expression.
Course of Biochemical and Cellular Technologies (Prof. Roberto Gambari):
The objectives of the course are the acquisition of theoretical principles and their application fields for the main biochemical and cellular methodologies applied in particular to the study of proteins and molecular and cellular interactions. The acquired knowledge includes: methods used to produce and purify proteins; analytical techniques used to analyze the amino acid composition, concentration, interactions with other biomolecules (nucleic acids, antibodies, transcription factors, etc.); the different cell cultures, culture, observation and analysis methods; the radioisotopes and their major applications in molecular and cell biology. Prerequisites
- Course of Molecular and Recombinant Technologies (Dr. Alessia Finotti):
Knowledge of Chemistry, Biology, Biochemistry and Molecular Biology.
Course of Biochemical and Cellular Technologies (Prof. Roberto Gambari):
Basic Knowledges of chemistry, biochemistry and molecular biology are required.
No prerequisites. Course programme
- Course of Molecular and Recombinant Technologies (Dr. Alessia Finotti):
40 hours of classroom didactic lecturing.
Topics addressed:
FIRST SECTION: Methodologies for the analysis of nucleic acids and their expression/ modulation (20 hours):
- Extraction and purification of DNA, quantification and analysis: electrophoresis using agarose and polyacrylamide gels, capillary electrophoresis;
- Techniques for genomic analysis: enzymes for DNA manipulation (e.g. restriction enzymes, polymerases, ligase), Southern blotting, Polymerase Chain Reaction and its variants, gene sequencing and Next Generation Sequencing;
- Extraction and purification of RNA and microRNAs;
-Assays for gene expression studies: reverse transcription, quantitative real-time PCR, digital PCR and gene expression arrays;
SECOND SECTION: Techniques of DNA modification, cloning vectors, expression vectors, transfection systems, recombinant proteins (20 hours):
- Enzymes for DNA cloning and basic principles of recombinant technologies;
- Molecular cloning vectors and their applications (plasmids, bacteriophages, cosmids, ecc.); genomic libraries production;
- Transfection and other methods of introducing foreign DNA into living cells;
- Cloning approaches for gene expression study and gene expression modulation: use of RNA interference and gene silencing;
- Assay for promoter and transcription factors analyses;
-Introduction to GeneTherapy vectors;
-Methods for recombinant protein production: vectors, expression systems for recombinant proteins, pharming, examples of therapeutic applications; - Monoclonal antibodies.
In addition to the 40 hours of lectures (5CFU), it will be held a laboratory exercise (1CFU) divided in a section of practical laboratory experience and a section of preparation and analysis of the results carried out using PowerPoint lectures, data analysis and exercises with the possible assistance of videos and animations. Principal topic: total RNA from biological matrices, its spectrophotometric quantification, qualitative and quantitative analysis of the RNA extracted by agarose gel electrophoresis.
Course of Biochemical and Cellular Technologies (Prof. Roberto Gambari):
48 hours of classroom didactic lecturing.
Topics addressed:
- Amino Acids and Proteins: from structure to function (2 hours).
- Isolation of cells from tissues. Flow cytofluorimetry; hemochromocytometric examination with leukocyte formula (4 hours).
- Chromatographic techniques: TLC, classical column liquid chromatography: normal phase or reverse phase, partition, with the exception of dimensional, ionic exchange, affinity, HPLC. Importance in biomedicine (6 hours).
- Protein purification methods (2 hours).
- Spectrophotometric techniques: description of the instrumentation. Theory behind spectrophotometry. Quantitative analysis of biological molecules (2 hours).
-Electrophoresis: cellulose acetate; polyacrylamide gel; SDS-PAGE; EMSA test; Footprinting; two-dimensional gels; capillary electrophoresis (8 hours).
- General aspects of the immune system; Polyclonal and monoclonal antibodies. Radioimmunoassay (RIA) assay. ELISA tests and applications. LUMINEX technology and applications. Western blotting technique: applications in cellular and molecular biology (8 hours)
- Biochemical technologies for the quantification and analysis of molecular interactions (Biosensors) (2 hours).
- Basic principles for working with cells and tissues: cell isolation; cell display; Eukaryotic cell lines; Immortalized and transformed cells. Storage and maintenance of cell cultures; cell treatments for drug tests (6 hours).
- Apoptosis and cell cycle: molecular biology of cancer (statistics, definition, characteristics) (4 hours).
- Cellular Biobanks (2 hours).
- Single cell analysis (2 hours).
The hours for the specific issues are indicative. Didactic methods
- Course of Molecular and Recombinant Technologies (Dr. Alessia Finotti):
The course is structured in 40 hours of classroom didactic lecturing.
During didactic lecture the course topics will be explained. In the classroom it’ll be also suggested scientific journals, websites, as references and explainer videos. The powerpoint presentation of the topics will be provided by the teacher after the lesson (PDF format).
Suggested books are recommended for a more simple understanding of the basic concepts, but they do not talk about all the arguments explained by teaching and learning in the classroom.
In addition to the 40 hours of lectures (5CFU), it will be held a laboratory exercise (1CFU) divided in a section of practical laboratory experience and a section of preparation and analysis of the results carried out using PowerPoint lectures, data analysis and exercises with the possible assistance of videos and animations.
For the practical laboratory exercise, the students will be divided into groups, and will work individually or in pairs, on these topics: the extraction of total RNA from biological matrices, its spectrophotometric quantification, qualitative and quantitative analysis of the RNA extracted by gel electrophoresis of agarose, retrotranscription and qPCR real time.
Course of Biochemical and Cellular Technologies (Prof. Roberto Gambari):
The course consists of frontal lectures (48 hours, 6 ECTS), weekly in the classroom using slides in power point format and blackboard provided in the classrooms to explain better the topics. Suggested books are recommended for a more simple understanding of the basic concepts, but they do not talk about all the arguments explained by teaching and learning in the classroom. Therefore, the attendance to the lessors is highly recommended. Learning assessment procedures
- Course of Molecular and Recombinant Technologies (Dr. Alessia Finotti):
The written exam includes 31 questions with multiple choice (with 5 answers only one of which is correct) and will be carried out OMR modules. The exam will last up to 60 minutes.
For DSA students, the test procedure will be evaluated individually.
To pass the exam (vote 18/30) it will be necessary to correctly answer at least 18 questions. The vote of 30/30 and honors will be achieved by answering correctly to 31 questions. No penalty points will be counted for wrong answers or unspecified answers.
Course of Biochemical and Cellular Technologies (Prof. Roberto Gambari):
The written exam includes 31 questions with multiple choice (with 5 answers only one of which is correct) and will be carried out OMR modules. The exam will last up to 60 minutes.
For DSA students, the test procedure will be evaluated individually.
To pass the exam (vote 18/30) it will be necessary to correctly answer at least 18 questions. The vote of 30/30 and honors will be achieved by answering correctly to 31 questions. No penalty points will be counted for wrong or unspecified answers.
The final vote of Biochemical and Recombinant Technologies (IC) is the average of the scores obtained in the two modules: Molecular and Recombinant Technologies (Dr. Alessia Finotti) and
Biochemical and Cellular Technologies (Prof. Roberto Gambari). In the case of intermediate average value, the final vote will be rounded up or down base on the attitude and performance in laboratory experiences. Reference texts
- Course of Molecular and Recombinant Technologies (Dr. Alessia Finotti):
Slides were shown during the frontal lectures (PDF format); other material provided by the teacher.
Principal suggested text:
-Biotecnologie Molecolari. Principi e tecniche. Terry A. Brown - ZANICHELLI
Any further information on the following texts:
-Metodologie biochimiche e biomolecolari. M. Maccarrone - ZANICHELLI
-Genomi 4. IV edizione, 2018. Terry A. Brown - 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
-Metodologie Biochimiche. Bonaccorsi di Patti, Contestabile, Di Salvo - ZANICHELLI
Course of Biochemical and Cellular Technologies (Prof. Roberto Gambari):
Slides shown during the frontal lectures (PDF format).
Specific topics can be further developed in the following texts, as indicated in the slides shown during the frontal lectures:
-Metodologie Biochimiche, Maria Carmela Bonaccorsi, Roberto Contestabile, Martino Di Salvo. Casa Editrice Ambrosiana. Distribuzione esclusiva Zanichelli (2012).
-Biochimica Applicata, ¿Monica Stoppini, Vittorio Bellotti. Edises (2012). ¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿
-Biologia molecolare della cellula, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter, Zanichelli Editore (2009).¿¿¿¿¿
-Metodologie biochimiche e biomolecolari. M. Maccarrone - ZANICHELLI
-Genomi 4. IV edizione, 2018. Terry A. Brown - EDISES
