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BIO732 : Gene Manipulation and Genetic Engineering

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Dr. Basharat Ali
University of the Punjab, Lahore

Course Contents

Introduction, What is genetic engineering, Genes and Genome, The flow of genetic information, Molecular cloning, Basic techniques, Basic Techniques of Gene Manipulation-an overview, Isolation of genomic DNA, Agarose gel electrophoresis, Nucleic acid blotting, Autoradiography, Southern blotting, Northern blotting, Western blotting, Transformation of E. coli , Electroporation, Transformation with other organisms, Polymerase Chain Reaction (PCR), Cutting and Joining DNA molecules, Host-controlled restriction modification, Types of restriction and modification (R-M) system, Nomenclature of restriction endonucleases, Target sites of restriction endonucleases, Number and size of restriction fragments, Summary of restriction endonucleases, DNA modifying enzymes, Methods of joining DNA fragments, DNA ligase to create covalent, recombinant DNA, Alkaline Phosphatase, Blunt end ligation via linker molecules, Adaptors, Homopolymer tailing, Cloning of cDNA by Homopolymer tailing, Plasmids, Interconversion of plasmid DNA, Effect of ethidium bromide on supercoiling of DNA, Phenotypic traits exhibited by plasmids, Properties of Conjugative and non-conjugative plasmids, Host range of plasmids, Partitioning and segregative stability of plasmids, Incompatibility of plasmids, Purification of plasmid DNA, Desirable properties of plasmid cloning vehicles, Natural plasmids as cloning vehicles, Use of pSC101 for cloning, pBR322, a purpose-built cloning vehicle, Cloning with pBR322, Improved vectors derived from pBR322, Runaway plasmid vectors, Bacteriophage lambda as cloning vector, Replication of phage-lambda DNA in lytic and lysogenic cycles, Modified lambda phages, Steps in cloning with lambda, Packaging phage-lambda DNA in vitro, Vectors for DNA sequencing: bacteriophage M13, Cosmid vectors, Modified schemes for cloning in Cosmid vectors, Phasmid vectors, Bacterial artificial chromosomes (BACs), Yeast artificial chromosomes (YACs), Shuttle and Expression vectors, Comparison of different cloning vectors-summary, Overview of cloning strategies, Genomic DNA libraries, LambdaEMBL vectors for genomic library construction, Genomic libraries in high-capacity vectors, PCR as an alternative to genomic DNA cloning, Properties of cDNA, cDNA libraries, Preparation of cDNA for cloning, Improved methods for cDNA cloning, PCR as an alternative for cDNA cloning, Screening strategies, Screening by hybridization, Benton and Davis’ plaque lift procedure, Probe design, Chromosome walking, Chromosome jumping, Screening by PCR, Expression cloning, Immunochemical screening, Immunochemical screening of lambdagt 11, South-western and north-western blotting, Screening by functional complementation, Requirement for expression in E. coli, Secretion of proteins, Protein trafficking, Stability of foreign proteins in E. coli, Constructing the optimal promoter, Optimizing translation initiation, Stability of mRNA and codon choice, The effect of plasmid copy number, Plasmid stability, Structural instability, Host cell physiology can affect the level of expression, DNA sequencing: Benefits and Applications, Maxam-Gilbert method, Chain termination or dideoxy procedure Modifications of chain terminator sequencing, Automated DNA sequencing, Sequencing accuracy, DNA sequence databases, Mutagenesis, Cassette mutagenesis, Primer extension: the single primer method, PCR methods for site-directed mutagenesis, Basic PCR reaction, PCR principles and procedure, DNA polymerases, Primers, Degenerate primers, Types of PCR, Competitor RT-PCR, Real-time quantitative PCR (qPCR), Nested PCR, Inverse PCR, Multiplex PCR, RAPD, RFLP, AFLP-PCR, Applications of PCR, PCR-Medicine, PCR-Forensic sciences, PCR-Agricultural sciences and environment, PCR-Molecular paleontology, Genome mapping, Markers for genome mapping, Genetic linkage mapping, Physical mapping, Physical versus linkage maps, The use of RFLPs in physical maps, STS in physical maps, SNPs as physical markers, Polymorphic DNA detection in the absence of sequence information, AFLPs detection in the absence of sequence information, Fluorescence in situ hybridization (FISH), Radiation hybrid (RH) mapping, Happy mapping, Map integration, Sequencing Genome, High-throughput sequencing, Shotgun sequencing, Clone-by-clone sequencing, Orthologs and paralogs, Comparative genomics of bacteria, Comparative genomics of organelles, Comparative genomics of eukaryotes, DNA Microarray, Spotted DNA Arrays, Oligonucleotide Chips, Applications of Microarray-Microbial gene expression analysis, Applications of Microarray-Profiling in human disease, Phage Display, Screening phage display libraries, Applications of phage display, Knock Outs & Knock Inns, siRNA Technology, Applications of siRNA, Proteomics, Two-dimensional (2-D) electrophoresis, Mass Spectrometry, Protein Microarrays, Cloning in gram –ve other than E. coli, Cloning in gram +ve bacteria B. subtilis, Cloning in Streptomyces, Cloning in Archaea, Cloning in S. cerevisiae, Transformation of fungi with exogenous DNA, Vectors for use in S. cerevisiae, Properties of different yeast vectors, Promoter system for yeast, Multipurpose vectors for use in yeast, Cloning of large DNA fragments, Deficiencies and advantages of YACs, Plant transformation, Callus culture, Cell suspension culture, Protoplast culture, Regenration of fertile plants, Major strategies for transformation, Agrobacterium mediated transformation, T-DNA transfer, Function of T-DNA gene transfer, Disarmed Ti vectors, Two plasmid strategy, Leaf disc transformation, Agrobacterium and monocots, Rhizogenes and Ri plasmids, Protoplast transformation, Particle bombardment, Chloroplast transformation, In planta transformation, Plant viruses as vectors, Genetic modification in animals, Strategies to transform animal cells, Chemical transfection techniques, Transfection with polyplexes, Transfection with liposomes and lipoplexes, Physical transfection techniques, Microinjections, Selectable markers for animal cell gene transfer, Bacterial vectors for animal transfection, Vrial vectors for transfection, Transgenic mice production, Pronuclear microinjection, Recombinant retroviruses, Transfection ES cells, Application of transgenic mice, Gene transfer to Xenopus, Gene transfer to Fish, Ways to exploit recombinant DNA, Production of recombinant proteins, Biopharmaceuticals approved, Transgenic animals and plants, Recombinant proteins in plants, Metabolic engineering in bacteria, Metabolic engineering in plants, Herbicide resistance in plants, Recombinant virus production, Fungal resistance, Resistance to bacteria, BT an insect resistance gene source, Drought resistance transgenic crops, Transgenic crops for poor quality soil, Biotechnology role in food, Transgenic model animals, Gene medicine, DNA vaccines, Gene augmentation therapy, Gene therapy in cancer, Ethics in gene manipulation.