Learning objectives
The course aims to introduce the basic molecular biology techniques used in the isolation and cloning of genes. <br />
Prerequisites
Students are required to have passed the Biochemistry and Molecular Biology exam
Course unit content
<br />1. Enzymes Restriction enzymes Methylation of DNA. Ligase DNA polymerase Reverse Transcriptase (RNA-dependent DNA polymerase). Terminal Transferase. RNA polymerase. Polynucleotide kinase of T4 phage. Alkaline Phosphatase. Nuclease <br />
2. Purification of DNA and RNA. Washing with Phenol, Phenol-Chloroform, Chloroform. Precipitation with alcohol and drying. Resuspension in TE or water. Determination of concentration and purity <br />
3. Bacteria Growth mediums Genotype Conservation and propagation <br />
4. Bacterial plasmids Replication and Incompatibility Mobility Selection Markers Development of plasmids as cloning vectors Extraction and purification of plasmid DNA from bacteria. Digestion with restriction enzymes. Purification of digestion products. Ligation. Strategies for ligation of an exogenous DNA in a plasmid vector. Transformation of bacteria. Identification of bacterial colonies containing recombinant plasmids. Identification of the recombinant of interest Possible results of a ligation + transformation. Checks on the ligation + transformation. Amplification and conservation of clones and plasmid libraries <br />
5. Bacteriophages lambda Molecular biology of the bacteriophage lambda - Construction of vectors based on the bacteriophage lambda Titration of bacteria Titration of phages Extraction and purification of phage DNA. Preparation of recombinant phages. Identification of plaques that contain recombinant phages. Amplification and conservation of clones or phage libraries <br />
6. Cosmids Characteristics of cosmids. Construction of genome libraries in cosmids. Amplification and conservation of clones or cosmid libraries <br />
7. Filamentous bacteriophages with a single filament. Molecular biology of filamentous bacteriophages Cloning in the intergenic region of the RF. Transformation and formation of plaques. Identification of plaques that contain recombinant phages. Extraction and purification of phage DNA. Multiplication of phages in liquid medium. Purification of phage DNA (PEG+ Phenol-Chloroform). Amplification and conservation of clones <br />
8. Phagemids Characteristics of Phagemids. Cloning in Phagemids. Production of single strand DNA by means of Helper Phage. Construction of acDNA libraries in phagemids lambdaZAP: characteristics Cloning and plasmid recovery <br />
9. Gel electrophoresis. Agarose gel electrophoresis. Recovery of DNA from the gel. Denaturing agarose gel (alkaline, formaldehyde). Electrophoresis on non-denaturing polyacrylamide gel. Electrophoresis on denaturing polyacrylamide gel. Autoradiography. Recovery of DNA from the gel. Analysis and cloning of eukaryotic genomic DNA. Isolation of eukaryotic genomic DNA. Fragmentation of genome DNA. Size of a genomic library. High capacity vectors used for genome libraries. Screening of a genomic library <br />
10. Extraction and purification of RNA. Preparation of the materials and solutions used in RNA extraction. Classic extraction methods. Separation of Poly(A)+RNA <br />
11. Construction and analysis of cDNA libraries. Size of a cDNA library. Synthesis of the first strand of cDNA. Synthesis of the second strand of cDNA. Cloning of cDNA in a vector. Screening by means of nucleic acid hybridation <br />
12. PCR. Essential components of PCR. Thermal phases of PCR. Design of primers. Contamination problem. Hot start. Analysis for gel electrophoresis. Cloning of amplicons. Nested PCR. Long PCR. Mutagenesis for PCR. Reverse Transcriptase-PCR (RT-PCR). Touchdown PCR <br />
13. Sequencing. Maxam and Gilbert method. Sanger method. Sequencing of long DNA fragments and entire genomes
Full programme
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Bibliography
Dai geni ai genomi - Dale, von Schantz - EdiSES <br />
Analisi dei geni e genomi - Richard J.Reece - EdiSES <br />
Ingegneria genetica. Principi e tecniche. S.Primrose et al. - Zanichelli <br />
DNA Ricombinante - J.D.Watson et al. - Zanichelli
Teaching methods
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Assessment methods and criteria
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Other information
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