MOLECULAR AND RECOMBINANT TECHNOLOGIES
Academic year and teacher
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- Versione italiana
- Academic year
- 2016/2017
- Teacher
- GIOVANNI BERNACCHIA
- Credits
- 12
- Curriculum
- BIOTECNOLOGIE PER L'AMBIENTE
- Didactic period
- Annuale
Training objectives
- This course is aimed at teaching the techniques and the basic methodologies to study and analyze nucleic acids and gene expression.
Molecular and Recombinant Technologies.
Recombinant DNA technology. The objectives are the improvement and broadening of the knowledge acquired of basic tools and thier applications in gene therapy.
The student will be guided towards understanding not only the methodologies and tools used in the field of recombinant molecular technologies, but we will try to transfer some knowledge of their applications, such as "tools" they need to use to obtain specific experimental goals.
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, 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.
KNOWLEDGE AND UNDERSTANDING
The student learns:
- the correct use of scientific terms
- the techniques used to purify nucleic acids
- the basal DNA sequencing methods, but also the basics of the next generation sequencig and its possible applications
- the basic elements to build a recombinant vector
- the techniques used to introduce an exogen DNA fragment in a cloning vector
- the different type of vectors
ABILITY TO APPLY KNOWLEDGE AND UNDERSTANDING
The student knows:
- the DNA, RNA extraction methods and analysis.
- the use of genomics and transcriptomics analyses.
- the techniques and the instrumentation used for single cell analysis
- which technique should be used for specific applications, such as production of DNA and cDNA library
- the suitable vectors used for protein production
- the vectors used to analyze and modify the expression of specific genes, such as their modulation by microRNA
- the recombinant vector suitable for gene therapy and transgenic organism production.
Plant Molecular Biology.
The course will provide basic and specific knowledge of the molecular mechanisms that regulate the functioning of the plant organisms, with particular attention to the phenomena that are unique in plants. These skills are described taking into consideration possible modifications and applications in agriculture and industry.
KNOWLEDGE AND UNDERSTANDING
The student will learn:
The structure of plant genomes (differences in size, composition and structure in monocot and dicot genomes)
Function and regulation of gene expression in plants (promoters and terminators, marker and reporter genes)
The methods for the genetic modification of plants (Agrobacterium tumefaciens and direct methods)
Some of the applications of transgenic plants and the related problems
The response mechanisms of higher plants to different types of stress (biotic or abiotic) and possible improvements
The mechanisms of gene silencing in plants
The basics of plant breeding and molecular breeding
ABILITY TO APPLY KNOWLEDGE AND UNDERSTANDING
The student will know how:
to use the correct terminology of plant molecular biology
to understand and manipulate the modular structure of plant gene
to design experimental strategies of study applied to plants of agricultural interest
to critically evaluate the experimental results. Prerequisites
- Molecular and Recombinant Technologies: Chemistry, Biology, Biochemistry and Molecular Biology.
Plant Molecular Biology: Plant Physiology and Molecular Biology.
Knowledge of molecular biology and plant physiology. Course programme
- Molecular and Recombinant Technologies.
The lessons raise two issues: methodologies for the analysis of nucleic acids and their expression and/or modulation (first section) and techniques of DNA manipulation, cloning vectors, transfection methods, and examples of applications in Biotechnology (second section)
FIRST SECTION: methodologies for the analysis of nucleic acids and their expression and/or modulation
Extraction and purification of DNA, quantification and analysis: electrophoresis using agarose and Polyacrylamide gels, capillary electrophoresis (2 hours).
Techniques for genomic analysis: the employment of enzymes (restriction enzymes, polymerases, and so on), southern blotting, Polimerase Chain Reaction, SNP-array, gene sequencing and Next Generation Sequencing (8 hours).
Extraction and purification of RNA and microRNA introduction; reverse transcription, quantitative real time PCR, digital PCR and gene expression array (7 hours)
Techniques for single-cell analysis: FACS, cDNA-PCR and array technology as RNA-sequencing, GCH array, Chip- and methyl-sequencing, (5 hours)
SECOND SECTION: Techniques of DNA modification, cloning vectors, transfection systems, and biotechnological applications
Extraction methods for the isolation of plasmid DNA, DNA modifying enzymes and bases of recombinant technologies (2 hours)
Molecular cloning vectors and their applications; library (4 hours)
Array technology for study of gene expression and modulation: use of RNA interference and gene silencing (4 hours)
Oligonucleotides used for cloning into expression vector to evaluate microRNA activity (2 hours)
Transfection and methods of introducing foreign DNA into an eukaryotic cell (2 hours)
Oligonucleotide-directed mutagenesis into an expression vector (1 hour)
Vectors in gene therapy, Genome Editing and CRISPR-Cas system, Restriction Modification system (2 hours)
Cloning (1 hour)
Plant Molecular Biology.
Lessons are structured in different topics, with increasing complexity, the normal duration is about 2 hours each.
Plant in vitro cultures and micropropagation, role of hormones and applications -- The plant gene, the role of the promoter and the terminator, structural modularity -- Methods for plant genetic transformation, Agrobacterium tumefaciens and direct methods, transformation vectors, working protocols -- Applications and issues associated with the production and growth of transgenic varieties -- gene expression regulation in plants, antisense RNA and RNA interference, applications -- organization, structure and characteristics of plant genomes, size, repetitive elements and variability -- genetic and physical maps, enrichment in molecular markers and molecular techniques for the identification of new markers – plant breeding in species of agronomic interest, classic and molecular methods for cross and selection – plant responses to stress and pathogens, new applications. Didactic methods
- The topics will be addressed in depth in the lessons, where references to scientific journals, websites treating the various topics and explanatory videos will be used. The topics will be explained through powerpoint presentations, that will provided to the students. The textbooks do not describe all the topics of the course (are mainly recommended for a better understanding of basic concepts), it is therefore recommended to participate at the lessons.
Learning assessment procedures
- Oral examination will take place at the end of the course and during other sessions. The exam is designed to test the level of knowledge and assay the depth-analysis of the topics and scientific reasoning skills developed by the student. The test consists of an interview of at least 30 minutes. The grade is expressed in thirtieth and the minimum grade is 18. The examination is passed, if the student demonstrates enough knowledge and reasoning skills. The final grade for the course Molecular and Recombinant Technologies (IC) is the average obtained in the two modules.
Molecular and Recombinant Technologies.
The exam usually starts with a topic chosen by the student. During the examination the student will be asked more in-depth questions about the contents of the two course sections, the questions gradually test the detailed understanding obtained by student. Positive result is obtained when the student gives good answers and demonstrates ability to apply the techniques described during the course. The test is failed if the student cannot answer anything about the general questions in one of the topics covered, or if during the exam the student does not show adequate understanding.
Plant Molecular Biology.
The student will respond to an average of three general questions, followed by more in-depth questions that may wander to other subjects of the course. It is particularly valued the ability to relate the different topics of the course and the ability to critically describe knowledge acquired. Reference texts
- Molecular and Recombinant Technologies.
Books (in italian):
-Biotecnologie Molecolari. Principi e tecniche. Prima edizione italiana condotta sulla quinta edizione inglese. Terry A. Brown - ZANICHELLI
-Ingegneria genetica. Principi e Tecniche. S. Primrose et al. - ZANICHELLI
-DNA ricombinante J.D. Watson et al. - ZANICHELLI
-Dai geni ai Genomi. Dale, von Schantz - EdiSES
-Analisi dei geni ai Genomi. Richerd J. Reece - EdiSES
-Teaching support materials
Plant Molecular Biology.
Updated articles and reviews taken from international journals. Notes and course material used during the lessons.
Buchanan B., Gruissem W., Jones R. Biochimica e Biologia Molecolare delle Piante. Ed. Zanichelli
