Syllabus
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Course Code: BT-201 Course Name: Genetic Engineering |
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| MODULE NO / UNIT | COURSE SYLLABUS CONTENTS OF MODULE | NOTES |
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| 1 | Genetic Engineering Introduction and scope of Genetic Engineering, Miles stones in Genetic engineering Nucleic Acids Purification of total cell DNA, plasmid DNA, phage DNA, Yield Analysis, , Nucleic acid blotting and hybridization Manipulation of purified DNA DNA modifying enzymes- Terminal deoxynucleotidyl transferase, Polynucleotide kinase, Alkaline phosphatase, Nucleases, Methylases Restriction Endonucleases- Host controlled restriction and modification, Nomenclature, types, Recognition sequence, blunt and sticky ends, applications. Ligases- E. coli and T4 DNA ligases, Linker, Adaptor, Homopolymer tailing Gene Cloning Vectors General features, Types of cloning vectors- Plasmid, bacteriophage, phagemid, cosmid, artificial chromosomes (YAC, BAC, PAC) |
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| 2 | Transformation of E. coli Concept, Selection of transformed cells, Identification of recombinants (bacteria and phages) Cloning of Specific Gene Direct selection, identification from a gene library-genomic library, cDNA synthesis and cloning-Properties of cDNA, mRNA enrichment, cDNA library. Methods for Clone Identification Screening strategies-Colony and plaque hybridization, Abundancy probing, Heterologous probing, Immunological screening, Differential screening, Subtractive hybridization. Protein-Protein Interactions-Phage display, Yeast two hybrid system, Yeast three hybrid system. |
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| 3 | Nucleic Acid Sequencing DNA Sequencing: Rapid DNA sequencing techniques and strategic details of range of methodologies e.g. Dideoxyribonucleotide chain termination, Chemical degradation, Automated DNA sequencing, Thermal cycle sequencing, Pyrosequencing. Polymerase Chain Reaction Concept, Basic PCR reaction, Factors affecting the PCR, Types of PCR (RT- PCR, Real time PCR, Allele specific PCR, Multiplex PCR) , Applications of PCR Site Directed Mutagenesis Oligonucleotide directed mutagenesis, PCR amplified oligonucleotide directed mutagenesis, Random mutagenesis with degenerate oligonucleotide primers / nucleotide analogs. |
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| 4 | Gene expression and Regulation studies Primer extension, S1 mapping, Gel retardation assay, Deletion analysis, Reporter genes, DNA foot printing, Modification interference assays, HRT, HART Manipulation of gene expression in prokaryotes Problems with production of recombinant proteins in E coli, optimizing expression of foreign genes in E. coli- Strong and regulatory promoters, Codon usage, Fusion proteins, Increasing protein stability and secretion, Translation expression vectors, Protease deficient host strains. Heterologous protein production in Eukaryotes Saccharomyces cerevisiae and Pistia pastoris expression systems, Baculovirus Insect cell expression systems, Mammalian cell expression system. |
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