Methods in Molecular Biology and Genetics (2020/2021)



Course code
4S008190
Credits
12
Coordinator
Antonella Furini
Academic sector
AGR/07 - AGRICULTURAL GENETICS
Language of instruction
Italian
Teaching is organised as follows:
Activity Credits Period Academic staff Timetable
teoria 6 II semestre, I semestre Linda Avesani, Diana Bellin, Antonella Furini, Sara Zenoni

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laboratorio [1° turno] 6 II semestre, I semestre Linda Avesani, Diana Bellin, Antonella Furini, Sara Zenoni

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laboratorio [2° turno] 6 II semestre, I semestre Linda Avesani, Diana Bellin, Antonella Furini, Sara Zenoni

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Learning outcomes

The course aims to provide basic knowledge of recombinant DNA technologies and methodologies applied to molecular genetic and genomic analysis. Frontal lessons will be introductory to traditional genetic methodologies and to the most advanced ones to analyze genes and their functions. Laboratory lessons will apply the background from frontal lessons by using the most common methodologies of molecular genetics used for prokaryotes and eukaryotes. At the end of the course the student will know the principal techniques related to the extraction, analysis and manipulation of nucleic acids; protein expression in heterologous systems; the techniques used for genetic transformation of animal and plant organisms; the methods for gene expression analysis and for studying protein-protein interactions. The acquired knowledge will enable the students to understand the methodological approaches used in scientific research in molecular genetics and to use molecular technologies for specific laboratory experiments.

Syllabus

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MM: teoria
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1) Cloning: - Plasmid vectors, selection markers and reporter genes (e.g. GFP); - E. coli transformation - Selection of recombinants - Methods of cloning: (i) Vectors based on restriction enzymes; (ii) PCR amplification and cloning of DNA fragments through T/A cloning: pGEM vectors; (iii) Cloning mediated by DNA topoisomerase, TOPO Cloning; (iv) Cloning by recombination: Gateway and Golden Gate Assembly systems; 2) Genetic transformation of plants (leaf disks and protoplasts) and animals (Prof. Furini). 3) Bacteriophage λ, Cosmids, BAC, YAC, genomic libraries and cDNA libraries, transformation of yeast cells; 4) Genomic DNA extraction, plasmid DNA extraction, phage DNA extraction; 5) DNA and genome analysis by hybridization (Southern blotting) and amplification, molecular markers; 6) Sanger DNA sequencing, NGS sequencing and third generation sequencing; genome sequencing and genomics; 7) Random and site specific mutagenesis; 8) Genome editing, CRISPR/Cas9; 9) Forward and reverse genetics to study gene function, gene silencing (Prof. Bellin). 10) RNA extraction and gene expression analysis by Northern blotting, RT-PCR and Real Time RT-PCR; 11) Large-scale expression analysis, transcriptomic platforms based on microarray and RNA-Seq; 12) Reporter genes and their use for functional studies and gene expression analysis; 13) DNA-protein interaction in the regulation of gene expression: chromatin immunoprecipitation (ChIP), shift assay, luciferase assay (Prof. Zenoni) 14) Sub-cellular targeting for recombinant protein production in heterologous systems (comparison between prokaryotic and eukaryotic systems); 15) Description of the systems used to study the sub-cellular localization of a protein; 16) Factors that influence the stability of a protein and its evaluation; 17) Techniques used for the quantification of solubilized proteins (western blot, ELISA, enzymatic assays (Prof. ssa Avesani).
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MM: laboratorio
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Preparation of constructs for plant and yeast transformation. Students will clone the GFP gene in a TOPO-cloning vector and subsequently into a Gateway vector by recombination: The gene of interest will be amplified by PCR and cloned in a vector thanks to the activity of the DNA isomerase I. From this construct (entry clone), the gene of interest will be transferred by recombination in two different Gateway vectors (destination vectors), for the expression in plant and yeast. During the procedure, recombinant constructs will be amplified in E. coli and purified by miniprep procedure. Final constructs will be utilized for genetic transformation of plants (Prof. Furini) and yeast in following experience. Genomic DNA analysis: genomic DNA extraction. Evaluation of DNA yield and quality based on spectrophotometric analysis and evaluation on agarose gel. Set up and optimization of SSR marker analysis on known and unknown genotypes: sample preparation for analysis on capillary electrophoresis sequencer for size determination, scoring of results. Example of parentage analysis, varietal identification and genomic analysis based on linkage (Prof. ssa Bellin). Site-specific mutagenesis: planning of the mutagenesis, primer design for mutagenesis, PCR amplification and phosphorylation, ligation and digestion with DpnI for mutagenized plasmid enrichment, transformation of competent cells, recovery and screening of mutant plasmids and validation by evaluation of activity of the expressed protein (Prof. ssa Bellin). Gene expression analysis in a transgenic organism by real-time RT-PCR: Extraction of RNA from the transgenic organism and from the wild type. Evaluation of the quality and quantity of the extracted RNA, treatment with DNAse, reverse transcription and real-time RT-PCR reaction. Evaluation of the different levels of expression of the transgene in the wild type and in the transgenic organism by calculating the deltadeltaCt. (Prof. ssa Zenoni). Total Soluble Protein extraction from leaf tissue and from yeast and quantification. Evaluation of the yields of GFP production in plant systems and in yeast by ELISA and by fluorimetric analysis. (Prof. ssa Avesani)

Assessment methods and criteria

The final evaluation will be performed by a written exam, including open and multiple-choice questions concerning both the theory and practical activities. The final score will be calculated from the weighted averages of each part, including the evaluation of the laboratory reports. The positive evaluation of the exam concerning the parts given in the first term will be mandatory for assessing the exam of the second term.

Reference books
Activity Author Title Publisher Year ISBN Note
teoria Terry A. Brown Biotecnologie molecolari (Edizione 2) Zanichelli 2017 978-88-08-32096-4
laboratorio Terry A. Brown Biotecnologie molecolari (Edizione 2) Zanichelli 2017 978-88-08-32096-4
laboratorio Terry A. Brown Biotecnologie molecolari (Edizione 2) Zanichelli 2017 978-88-08-32096-4