Learning objectives
The course aims to introduce the basic molecular biology techniques used in gene isolation and cloning, and in expression of recombinant proteins in a bacterial host
Prerequisites
No
Course unit content
1. Restriction enzymes • Methylation of DNA • DNA ligase • DNA polymerase • Reverse transcriptase (RNA dependent DNA polymerase) • Terminal Transferase • T4 Polynucleotide kinase • RNA polymerase • Alkaline phosphatase • Nuclease
2. Purification of DNA and RNA • Extraction with Phenol, Phenol-Chloroform, Chloroform • DNA precipitation with alcohol and drying • DNA resuspension in TE or water • Determination of the DNA concentration and purity
3. Bacterial growth • Media • Genotype • Conservation and propagation of bacteria
4. Bacterial plasmids • Replication and Incompatibility • Mobility • Selection Markers • Development of plasmids as cloning vectors and purification • Extraction of plasmid DNA from bacteria • Digestion with restriction enzymes • Purification of the digestion products • Strategies of ligation • Ligation of exogenous DNA into a plasmid vector • Bacterial Transformation • Identification of bacterial colonies containing recombinant plasmid of interest • Identification of possible results of ligation and transformation • Controls on ligation and transformation • Amplification and storage of clones and plasmid libraries
5. Bacteriophage lambda • Molecular biology of bacteriophage lambda • Construction of vectors based on bacteriophage lambda •Titration of bacteria • Titration of phages • Extraction and purification of phage DNA • Preparation of recombinant phage • Infection and plaques isolation • Identification of recombinant phages • Amplification and storage of clones or phage libraries
6. Characteristics of cosmid • Construction of cosmid genomic libraries • Amplification and storage of cosmid clones or cosmid libraries
7. Single-stranded filamentous bacteriophages • Molecular biology of filamentous bacteriophages • Cloning in the intergenic region of the RF • Processing and plaque formation • Identification of plaques containing recombinant phage • Extraction and purification of phage DNA • Multiplication of phage in liquid media • DNA Purification from phage • Amplification and conservation of the phage clones
8. Phagemids • Phagemid features • Cloning in the phagemid • Production of single-stranded DNA by helper phage • Construction of cDNA libraries in phagemid • lambda-ZAP • Cloning and recovery of the plasmid
9. Gel electrophoresis • Agarose gel electrophoresis • Recovery of DNA from gels • Denaturing agarose gel (alkaline, formaldehyde) • Electrophoresis on non-denaturing and denaturing polyacrylamide gel • Autoradiography • Recovery of DNA from the gel. • Analysis and cloning of eukaryotic genomic DNA • Isolation of genomic DNA • DNA Fragmentation of eukaryotic genomic • Complexity of a genomic library • High capacity vectors used for genomic libraries • Screening of a genomic library
10. Extraction and purification of RNA • Preparation of materials and solutions used in the RNA extraction • Traditional methods of extraction • Separation of Poly (A) + RNA
11. Construction and analysis of cDNA libraries • Complexity of a cDNA library • Synthesis of the first strand of cDNA • Synthesis of the second strand of the cDNA • Cloning of cDNA into a vector • Screening by hybridization of nucleic acids
12. PCR • Essential components of PCR • PCR thermal Phases • Design of primers • Problem of contamination • Hot Start • Analysis with gel electrophoresis • Cloning • Nested PCR • Amplicon • • Long PCR mutagenesis • Reverse Transcriptase-PCR (RT-PCR) • Touchdown PCR
13. Sequencing method of Maxam and Gilbert • Sequencing method of Sanger • sequencing of long DNA fragments and whole genomes
14. Expression in bacteria of a recombinant protein
Full programme
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Bibliography
Dai geni ai genomi - Dale, von Schantz - EdiSES
Analisi dei geni e genomi - Richard J.Reece - EdiSES
Ingegneria genetica. Principi e tecniche. S.Primrose et al. - Zanichelli
DNA Ricombinante - J.D.Watson et al. - Zanichelli
Teaching methods
Lecture
Assessment methods and criteria
Written examination
Other information
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