MOLECULAR TECHNOLOGIES
Academic year and teacher
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- Versione italiana
- Academic year
- 2017/2018
- Teacher
- FRANCO PAGANI
- Credits
- 6
- Curriculum
- Biologia molecolare e cellulare
- Didactic period
- Primo Semestre
- SSD
- BIO/11
Training objectives
- Educational goals
Through the acquisition of appropriate technical-molecular knowledge, the main educational goal of the course is to develop a scientific competence aimed at understanding and evaluating in a critical manner the recent scientific studies with particular emphasis to those connected with the pathophysiology of RNA. For each subject taken into account, the course will provide both the basic knowledge and the technical-molecular methodologies. This will be the base for understanding experiments and correctly evaluate the scientific aspects treated.
Knowledge and Understanding
The student knows:
- Biochemical and physiological bases of systems and cell biological processes
-chemical and biochemical basis of protein macromolecular structures and nucleic acids
- the basic cellular organization
-the basic techniques in biochemical and biological field and for the analysis of nucleic acids
Applying knowledge and understanding
The student is able to:
- Make correct use terminology related to nucleic acids
-understand the main metabolic pathways of nucleic acids Prerequisites
- The student must have basic knowledge of exact sciences (physics,
chemistry and mathematics) and a knowledge of cell biology and
biochemistry. Course programme
- The course consists of frontal lectures. The course will be mainly oriented on RNA processing in eukaryotes with particular reference to splicing, polyadenylation and miRNAs biosynthesis. It will also consider examples of human diseases caused by defects in RNA processing and technologies for the development of mouse models. The final examination will be a written essay.
The course will also include Journal Clubs prepared by the students.
1) pre-mRNA splicing: regulation and basic aspects (10h)
- splicing signals canonical splice sites, branch sites and site polyadenylazione. Introns GT-AG and AT-AC.
- the biochemistry of splicing
- the spliceosome and the role of snRNPs
- the non-canonical splicing signals (Enhancer and Silencer, ESE , ESS , ISE and ISS )
- splicing factors SR proteins , hnRNP and tissue-specific factors (NOVA , FOX, PTB)
- alternative splicing (basic mechanisms in the regulation of alternative splicing )
2) pre-mRNA splicing: basic technologies (8h)
- Methods for the analysis of splicing : splicing in vitro and analysis
with the minigenes
-methods for analyzing snRNP particles: Elettromobility shift assay (EMSA) , EMSA and supershift, affinity purification and Rna Immuno Precipitation (RIP)
- methods for analyzing RNA - protein interactions in splicing
- The method of classical SELEX and SELEX functional
- CLIP-high throughoutput analysis of RNA protein interactions ( recent examples NOVA , FOX, PTB )
- Bioinformatics Analysis of splicing (splicing sites for programs - such as the fruitfly - , ESE finder for SR proteins and RESCUE ESE)
- the trans- splicing in the trypanosome and Smart ( spliceosomal mediated RNA trans-splicing , such as hemophilia A)
3) Capping and polyadenilation (4h)
- the capping of the messengers ( 5 ' RACE)
- The process of polyadenylation and analysis techniques (Northern , 3 ' RACE)
- Editing of RNA
4) Co- transcriptional RNA processsing (4h)
- the regulation of the biosynthesis of the pre cotranscrizionale mRNA (the role of the C-terminus of PolII and the Bentley experiments), effect of the promoter.
- RNA processing and chromatin ( epigenetic regulation of alternative splicing )
- The Nonsense Mediated Decay (NMD ) .
- Role of Alu repeats in the expansion of exons in primates : the concept of Alu exonization
4) Methods of RNA analysis (4h)
- RT-PCR, RNase protection, RNA synthesis in vitro by phage RNA polymerase , nuclear RUN ON, primer extension , Northern, RACE, CLIP
5) Molecular pathology of splicing (6h)
- effect of genomic mutations on splicing
- The spinal muscular atrophy (SMA) and the regulation of the SMN gene
- sequence repeat expansion diseases ( myotonic dystrophy , CUG repeats , RNA gain of function )
- Splicing mutations in cystic fibrosis
- concepts of splicing therapy (oligonucleotides in DMD and SMA)
6) miRNAs , biological function and methods of analysis (4h)
- miRNA biosynthesis and processing
- miRNA biological effect
- the Mirtron
- siRNAs
- the dual luciferase assay for miRNAs , Northern blot for miRNAs
-siRNA: methods for silencing of endogenous genes oligonucleotides and plasmids
- alternative pathways in the processing of miRNAs
- Transcription and miRNA Gene Silencing
7) Technical aspects in murine models (4h)
- ENU mutagenesis
- Gene trapping
- creation of the classic transgenic mouse
- KO mice
- systems TetON Tet OFF
- conditional KO mice (cre recombinase and related technology)
- Example of a conditional mouse fibronectin
8) Novel DNA editing technologies (4h)
-CRISP CAS 9
-TALE
-ZINC FINGERS
- The Zinc Finger Nucleases and the CRISP in the creation of novel mouse models and in gene therapy Didactic methods
- The course will consists of frontal lessons. In particular, the 48 total
hours of teaching (6 credits). Classes are held weekly in the
classroom and the exposure will be done through the use of power
point slides and, for better understanding of the lessons even using
the classical blackboard. Learning assessment procedures
- Learning Assessment Procedures
The aim of the examination is to test the level of knowledge and depth of the topics covered during the course of the program of the course as well as 'the capacity' of reasoning developed by the student. The assessment is expressed in thirtieths (minimum grade 18). With the exception of students who have difficulties, the exam consists of a written examination with the administration of three open questions. As part of the topics covered during the current year, a question will deal with basic knowledge, a question about the technical aspects and the third will focus on a critical analysis of part of a scientific article. The answers to the questions are judged both for the content and for the appropriateness of the scientific language used. The maximum score is 10 points for each question Reference texts
- Schemes, figures and scientific articles (both original articles that review) provided by the teacher.
Molecular Biology of the Gene Watson Baker et al Zanichelli
