SyllabusThe subject will cover the following topics:
The syllabus will cover the following topics:
* review of basic molecular biology: theory, cloning vectors, mechanism of propagation;
* tools of genetic manipulation: restriction endonucleases, methylases, nucleases, polymerases, ribonucleases, exonucleases, transcriptases, ligases, kinases. Uses of primers, adaptors, linkers;
* cloning: cleavage of vector and insert, site compatibility, dephosphorylation of ends, ligation protocols and diagnostic techniques;
* preparation of transformed cells: transformation and transfection techniques, routine screening procedures;
* gene probing: hybridisation methods. Applications in biotechnology;
* strategies for gene isolation: cloning DNA libraries and genomic DNA libraries, screening for genes of interest by hybridisation and immuno-detection methods. Secondary verification using slot-blots, zoo-blots, Northern and Southern hybridisation. Final confirmation techniques: DNA sequencing and translation, homology searches, in vitro translation, hybrid arrest and release translation, immunoprecipitation and Western blot analysis;
* DNA fingerprinting: DNA profiling in forensic science. Ethics of human gene screening
* Expression engineering: promoters, enhancers, homeo-boxes, fundamental regulatory elements and empiricism in their juxtaposition. Secretion of engineered gene products; major factors affecting viability - refractile bodies, post-translational modification. Gearing up of production: The Eco-Sec(tm) System. Production, purification and analysis. Perspectives: development of expression vectors for mammalian cells; immunogenicity of engineered proteins - risk assessment in therapy;
* DNA (Phosphite triester) and Peptide Synthesis, protein engineering, oligonucleotides, peptide pharmaceuticals and automated synthesis. In vitro mutagenesis and elucidation of gene function
*Genetic therapy: human germ-line and somatic gene therapy; transgenic livestock for industry; transgenic animals as pharmaceutical factories
* DNA and RNA Sequencing: Maxam and Gilbert, Sanger dideoxy, M13 and plasmid/ supercoil-seqencing strategies, troubleshooting, direct genomic sequencing with polymerase chain reaction. Perspectives: The Genome Sequencing Project; automated sequenators;
* Scaling up mammalian and bacterial expression cultures: fermentation systems: batch, continuous and fed batch systems, turbidostat and chemostat cultures, downstream processing, inclusion bodies, secreted peptides, animal cell culture technology;
* applications in diagnostics, forensics medicine and agriculture: peptide vaccines, peptide pharmaceuticals, vaccinia viruses, antibiotics, recombinant vaccines, food and beverage improvement.
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