MOLECULAR BIOLOGY AND LABORATORY
The course explains the molecular mechanisms of information management in prokaryotic and eukaryotic cells. The acquisition of knowledge on this topic is necessary to understand the multiple functions of living beings, the dynamic of their evolution, the basis of pathology and the molecular keys of their adaptation to the different environments on the planet. The course is completed with the theoretical and practical study of the main investigation techniques for genomes and related transcripts
The student acquires the awareness that: a) the main molecules that participate in the thousands of chemical reactions, which occur simultaneously in a living being, are not numerous; b) all biological processes have a molecular basis, whose knowledge allows us to formulate understandable and reproducible models of universal value with the decisive use of modern information technology
AIMS AND LEARNING OUTCOMES
The course has the specific purpose of allowing the student to acquire the basic knowledge on on molecular biology. The acquisition of the required credits will be accomplished with the demonstration of an in-depth knowledge of the mechanisms underlying the processes of replication, transcription and translation of genetic information, as well as detailed information on the structural organization of nucleic acids, mechanisms of recombination and transposition and on the multiple functions of RNA. The students will also possess a clear and in-depth knowledge of the role of the genetic code as a universal information management system in living beings. The course will also enable the acquisition of information relating to the main techniques currently used for the study of the genome, such as PCR and quantitative PCR, nucleic acid sequencing and DNA chip and their relative applications in various professional fields (research, medical diagnostics, forensic medicine, monitoring of environmental and food quality) in addition to the basic techniques for the creation of libraries, cDNA library and the main techniques for recombinant DNA management The expected practical activity will also make the student able to autonomously manage the main laboratory techniques currently employed for the extraction and electrophoretic analysis of DNA, for obtaining cDNA from messengers extracted from eukaryotic cells, for their amplification to PCR medium and for the subsequent electrophoretic analysis of the results.
To approach in the most profiteable way the study of molecular biology, it is necessary to have acquired the knowledge of general and inorganic chemistry (in particular the basic concepts of chemical thermodynamics, chemical equilibria, pH), of organic chemistry (in particular a strong knowledge of the main classes of organic molecules and in-depth knowledge of the four classes of biological macromolecules) and biochemistry (in particular it requires a thorough knowledge of the structure of proteins, of the concept of enzymatic catalysis, of the main processes of metabolism).
Lectures and seminars
DNA structure. Gene concept. Chromatin and histones, the nucleosome and topoisomerases Molecular mechanisms of DNA replication in prokaryotes and eukaryotes. The DNA polymerases of prokaryotes and eukaryotes (structure, functions, nuclease activity), DNA primase, helicase. The control of fidelity of replication. The Okazaki fragments, the RNAse H; the DNA ligase. The mechanisms of beginning of replication in prokaryotes and eukaryotes, DNA A, the Replicator, the initiator, the role of CDk in controlling the beginning of replication. The end of replication in prokaryotes and eukaryotes; topoisomerase in bacteria and telomerase in eukaryotes. Transcription in prokaryotes and eukaryotes: promoters; transcription factors in bacteria; the RNA polymerases of prokaryotes and eukaryotes; the moderator; the termination of the transcription; the overall management of transcription in living beings. The maturation of the messenger in eukaryotes; capping in 5 ’, polyadenylation; autosplicing of type I and II introns; splicing managed by the spliceosome; alternative splicing The rRNA; tRNA synthesis. The synthesis of aminoacyl tRNA and aminoacyl tRNA synthetases. The genetic code. Translation in prokaryotes and eukaryotes. Post-transcriptional regulation of gene expression, regulatory RNAs in bacteria; riboswitches, RNAi, the molecular basis of RNA interference; the micro RNA. Specific site recombination and recombinases. VDJ recombination. Homologous recombination. DNA transposons. Retrotransposons. The SINES and the demonstration of their origin from retrotransposons. The synthesis of oligonucleotides. PCR, Principles and applications. The reverse transcriptase; the cDNA; amplification of the messenger; 3' and 5' RACE; Quantitative PCR. The analysis of the transcript: the DNA chips - construction and use techniques. Sequencing with the Sanger method; pyrosequencing, the 454 system, sequencing with the Illumina method. Restriction enzymes, restriction maps Recombinant DNA: basic principles, the different vectors (plasmids, phages, cosmids, BACs and YACs); how to build a library; CDNA libraries and their uses. Specific site mutagenesis. Sequencing of the human genome, techniques and problems. Satellite DNA and its uses in forensic genetics. DNA test and paternity test. Mitochondrial DNA and chloroplast DNA: replication, functional and structural characteristics. The use of mitochondrial DNA in the study of evolution. Mitochondrial Eve. Y-Adam. The use of mitochondrial DNA in forensic genetics. Synthetic biology. The definition of a minimal bacterial genome; the realization of the first artificial microorganism. The antisense approach. Antisense oligonucleoids; the PNAs Xenobiology: The expansion of the genetic code. The use of the Amber codon for the insertion of non-natural amino acids in proteins; orthogonal tRNAs and related synthetases Orthogonal ribosomes; the production of alien proteins; the XNAs, the XNA-DNA relationship Advanced investigation techniques of biological macromolecules: mass spectrometry (general principles and notes on specific applications) Nanotechnologies based on the use and manipulation of DNA. DNA origami; optical tweezers. The molecular basis of the origin of life. Practical part (1CFU): Plasmid DNA extraction from bacterial cells, digestion with restriction enzymes, agarose gel electrophoresis. RNA extraction from mammalian cell lines, RNA assay, cDNA retro-transcription. PCR amplification of a specific transcribed gene sequence. Electrophoretic analysis of the product
The in depth analysis of the contents of the program can be carried out on any university textbook on molecular biology offered by the market, as long as they are editions updated to the last five years.
Lectures and seminars
beginning of October
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The evaluation of the student will take place through a written verification of the knowledge and skills acquired during the practical part (max 3/30) and a final oral examination (max 28/30). The final score will be the addition of the two results (maximum 30 com laude/30)
The final evaluation will be carried out through oral examination giving a maximum score of 28/30, to which the score previously acquired in the practical part (max 3/30) will be added. The oral examination, for each student, requires to be examined by at least two members of the commission and to answer at least three questions concerning the topics listed in the program. In order to pass the exam, the student must demonstrate that he / she has acquired at least a sufficient knowledge of each of the three topics covered. The final evaluation will take into account the level of knowledge of the contents as well as the expository and reasoning skills demonstrated in the discussion conducted on the required topics. The final score will be formed by the addition of oral result to the score of the laboratory test. The latter will consist of a test carried out during class time concerning the verification of the theorical topics for laboratory activities and in a table where each student will have to enter the experimental results and in a final open test concerning the methods and the techniques used. The evaluation will contribute to the final vote according to the following criteria: test: up to a maximum of 1.5 points (from -1 to 1.5); laboratory results and relative final test: up to a maximum of 1.5 points (from -1 to 1.5). Maximum total score with laboratory activities: 3/30. In the event of a negative score, an additional oral examination will be carried out on the specific topics of the laboratory during the examination (the passing of the same does not include the assignment of points, the non-passing will cause the need to repeat the exam).