TECHNOLOGY OF RECOMBINANT DNA
Learning outcomes of the course unit
At the end of the course, the student should have learned the basic techniques to manipulate in vitro the three main biological macromolecules: DNA, RNA and Proteins. The course also aims to teach the various strategies used in gene cloning in microorganisms and in the expression of recombinant proteins in bacteria.
In particular, the student should be able to:
D1. Understand and learn the methodologies for amplifying the three main classes of biological macromolecules: DNA, RNA and Proteins. Learn the composition and replication methods of different types of bacterial vectors. Understand the chemical and physical mechanisms of the recombinant technologies. (Knowledge and understanding)
D2. Design the most correct strategy to achieve gene cloning within a bacterial vector. Define the analytical methodologies to check for the gene cloning, and establish possible control experiments. (Applying knowledge and understanding)
D3. Know how to evaluate the best methodology to perform gene cloning or the expression of recombinant proteins. (Independent judgment)
D5. Expose the results of an experiment of recombinant technologies even to an unqualified public. (Communication skills)
D5. Know how to connect the molecular mechanisms learned during the courses of Biochemistry and Molecular Biology with the application methods of recombinant DNA. Update on innovative techniques by consulting scientific publications. (Learning ability)
Course contents summary
The course will address the following topics:
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
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
The course will consist of lectures with the use of projected slides. During the course, exercises will be proposed, in which the students will be able to apply various procedures of Recombinant DNA Technologies. Educational material and examples of exercises will be made available on the Elly platform.
Assessment methods and criteria
The methods for verifying the level of knowledge of the student is a written exam consisting of 5 exercises / open questions. Students can view the exam by appointment with the teacher.
The mark will be calculated as the arithmetic average of the marks obtained in the individual exercises/open questions.