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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 to Gene Manipulation and Genetic Engineering, Flow of genetic information, Basic concepts of gene and genome, Molecular cloning, Schematic illustration of DNA cloning, 


Basic Techniques Of Gene Manipulation, Isolation of genomic DNA, Handling and quantification of DNA, Agarose gel electrophoresis, Polyacrylamide gel electrophoresis, Capillary blotting apparatus, Nucleic acid hybridization on membranes, Stringency control, Nucleic Acid blotting, Southern blotting, Northern blotting, Western blotting, Transformation of E. coli, Transformation of B. subtilis, Electrophoration,

CUTTING DNA MOLECULES, Restriction and modification of DNA, Restriction enzymes, Modification: Protection from restriction, Different restriction enzyme systems in one host, Restriction enzyme analysis of DNA, Host controlled restriction and modification, Page propagation on E. coli K or C Types of restriction and modification system, Characteristics of restriction endonucleases, Nomenclature of restriction of enzymes, Cleavage pattern of some restriction endonucleases Target sites, Affects of G+C content of DNA on its susceptibility to restriction endonucleases , Importance of elimination of restriction system in E. coli, Mechanical shearing of DNA,

JOINING DNA MOLECULES, DNA modifying enzymes, Polymerases Nucleases, Ligases, Reverse transcriptases, Diagrammatic explanation of Joining DNA termini by DNA ligase, Application of alkaline phosphatase, T4 DNA ligase-Blunt end ligation, Double linkers, Adaptors, Use of BamH1 adaptor molecule, Homopolymer tailing, Cloning cDNA by Homopolymer tailing, Use of Calf thymus terminal deoxynucleotidyl-transferase in joining DNA molecules, Diagrammatic explanation of synthesis of cDNA,

PLASMIDS-CLONING VEHICLES FOR USE IN E. COLI, Basic properties of plasmids Interconversion  of plasmid DNA, Effect of ethidium bromide on supercoiling of DNA, Phenotypic traits exhibited by plasmid- carried genes, Properties of conjugative and non-conjugative plasmids of gram-negative bacteria, Purification of plasmid DNA, Desirable properties of plasmids, Natural plasmids as cloning vehicles, Properties of natural plasmids used for cloning, Use of pSC101 for cloning, pBR322-a purpose building vehicles, Examples of the use of plasmid pBR322, Improved vectors derived from pBR322, Low copy number plasmids, Runaway plasmid vectors, Partitioning and segregative stability of plasmids,

BACTERIOPHAGE ʎ AS CLONING VECTOR, Replication of phage ʎ DNA in lytic and lysogenic cycles, Insertional and replacement vectors, Improved phage ʎ vectors, Packaging page ʎ DNA in vitro Scheme showing packaging of ʎ DNA into phage particles, Cosmid vectors, Phasmid vectors, cloning with single stranded DNA vectors, M13 phages, Bacterial artificial chromosomes (BACs), Yeast artificial chromosomes (YACs),

CLONING STRATEGIES-GENOMIC DNA LIBRARIES, Generalized scheme for DNA cloning in E. coli, Chromosome jumping strategies, Jumping library construction, cDNA, cDNA cloning, Full-length duplex cDNA synthesis,

SCREENING STRATEGIES, Genetic methods-selection for the presence of vector, Selection for inserted sequences, Immunochemical methods, Immunological screening of ʎgt11 or ʎZAP recombinant plaques, South-Western screening for DNA binding protein, Nucleic acid hybridization methods, Detection of recombinant clones by colony hybridization, Denton and Davis plaque-lift procedure, Differential screening, Diagrammatic explanation of differential screening, Chromosome Walking, Diagrammatic explanation of chromosome walking, Chromosome jumping, Expression of E. coli of cloned DNA molecules, Stability of foreign proteins in E. coli, Detection of expression of cloned genes, Maximizing the expression of cloned genes, Construction of the optimal promoter, Optimizing translation initiation, Stability of mRNA, The effect of plasmid copy number, Transcription termination, Plasmid stability, Structural stability of plasmids, Affect of host physiology on gene expression,

ANALYSIS OF DNA SEQUENCES, DNA sequencing by the Maxam Gilbert method, Reagents for Maxam and Gilbert DNA sequencing, Maxam and Gilbert-Autoradiography figure , Sequencing by the chain termination-dideoxy procedure, DNA sequencing with dideoxynucleoside triphosphates as chain terminators, Autoradiograph of chain terminator DNA, Automated sequencing,

POLYMERASE CHAIN REACTION (PCR)-HISTORY, PCR principles and procedure, Taq DNA polymerase, Primers, Degenerate primers, Types of PCR, Multiplex PCR, Nested PCR, RT-PCR, qPCR, Random amplified polymorphic DNA (RAPD), Restriction fragment length polymorphism (RFLP), Amplified fragment length polymorphism (AFLP), Applications of PCR-in Forensic, PCR-diagnosis, PCR-in study of evolution, PCR-Fossils, PCR-Gene cloning and expression, PCR-creating mutations, PCR-detection of pathogens,

FUNCTIONAL GENOMICS AND PROTEOMICS, Microarrays, Types of Microarrays, Phage display, Application of phage display, Knock outs, Knock ins, siRNA technology, Applications of siRNA technology, Protein expression analysis, Technologies for separation of protein in proteomics, 

SITE DIRECTED MUTAGENESIS, Cassette mutagenesis, Primer extension: the single primer method, PCR-method for site directed mutagenesis,

MANIPULATING DNA IN MICROBES, PLANTS AND ANIMALS CLONING IN GRAM –VE OTHER THAN E. COLI, Cloning in Gram +ve bacteria, Stability of cloning vector in Bacillus subtilis, Properties of S. aureus plasmids used as vectors in B. subtilis, Secretion of foreign proteins from B. subtilis, Cloning in Streptomyces, Vectors used with Streptomyces, Cloning in Archea,

CLONING IN SACCHAROMYCES CEREVISIAE AND OTHER FUNGI, Transformation of fungi with exogenous DNA, Vectors for use in S. cerevisiae, Yeast episomal plasmids, Yeast replicating plasmid, Properties of different yeast vectors, Promoter system for over expression of recombinant protein in yeast, Multipurpose vectors for use in yeast, Cloning with YAC, Deficiencies of classical YACs, Advantages of circular YACs over classical YACs, Gene Transfer to Plants, Plant tissue culture for transformation procedures,

STRATEGIES FOR GENE TRANSFER TO PLANT CELLS, Agrobacterium and plant interactions: Crown gall and hairy roots, Recognition of Agrobacterium by plant, Plasmid transfer and tumorigenesis: vir genes and T-DNA , Transfer of DNA to plant cells by Agrobacterium-Figure, Genetic Engineering with Ti plasmid,

AGROBACTERIUM MEDIATED TRANSFORMATION, Ti plasmid of Agrobacterium , T-DNA of Ti plasmid, Functions of T-DNA genes in A. tumefaciens Ti plasmids, Binary vectors, A two plasmid strategy to create a recombinant plant, A tobacco plant expressing the gene for firefly luciferase, Agrobacterium –mediated transformation of dicots, Leaf-disk  transformation by A. tumefaciens, Transformation of monocots, Transfer of large DNA segments in plants by using binary vectors, Agrobacterium rhizogenes transformation of plant roots,

DIRECT DNA TRANSFER TO PLANTS, Regeneration from transformed protoplast, Particle bombardment to transform plants, Direct DNA transfer to chloroplast, Plant viruses can be used as episomal expression vectors,

GENE TRANSFER TO ANIMAL CELLS, Major strategies for gene transfer to animal cells, Chemical transfection techniques-Calcium phosphate method, Transfection with polyplexis, Transfection with liposomes and lipoplexes, Physical transfection techniques-Electrophoration and ultrasound, Microinjection-Direct transfer of DNA, Selectable markers for animal cells-Endogenous markers, Plasmid vectors for the transfection of animal cell, Viruses as vector for gene transfer,

GENETIC MANIPULATION OF ANIMALS, Methods for the production of transgenic mice, Pronuclear microinjection, Recombinant retroviruses-germline transformation, Transfection of ES cells, Gene transfer to Xenopus, Study of in vitro cell free protein synthesis systems (CFPS), Applications and limitations of CFPS, Wheat germ S-30, Rabbit reticulocytes, Frog oocyte system,

APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY, Theme 1: Producing useful molecules, The different ways that recombinant DNA technology has been exploited, Commercial production of recombinant therapeutic proteins, Biopharmaceuticals approved in different countries, Use of transgenic animals and plants to produce recombinant proteins, Therapeutic recombinant proteins expressed in genetically modified plants, Metabolic engineering-production of small molecules in bacteria, Metabolic engineering-in plant to produce diverse chemical structures, Theme 2: Improving agronomic traits by genetic modification, Herbicide resistance in commercial transgenic plants, Production of virus resistance crops, Resistance against fungal pathogens, Resistance to bacterial diseases, Bacillus thuringiensis-source of insect resistance genes, Drought resistant transgenic crops, Transgenic plants to cope poor soil quality, Plant biotechnology to increase food yields, Theme 3: Using genetic modification to study, prevent and cure disease, Transgenic animals as model of human disease, Gene medicine to prevent, treat, or cure disease, DNA vaccines, Gene augmentation therapy, Gene therapy strategies for cancer,

ETHICAL CONCERNS OF GENETIC ENGINEERING, Biosafety and risks of genetically engineered products to humans, Impact on environment