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Gene Manipulation and Genetic Engineering (BIO732)

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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, FUNDAMENTALS TECHNIQUES OF GENE MANIPULATION-AN OVERVIEW, 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

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Course Codes

ACC311 ACC501 BIF401 BIF402 BIF601 BIF731 BIF732 BIF733 BIO101 BIO102 BIO201 BIO202 BIO203 BIO204 BIO301 BIO302 BIO303 BIO502 BIO731 BIO732 BIO733 BIO734 BIT701 BIT703 BIT710 BIT715 BNK601 BNK603 BNK604 BNK610 BNK611 BNK612 BNK613 BNK701 BNK703 BNK704 BNK725 BT101 BT102 BT301 BT302 BT501 BT603 BT605 BT731 BT732 BT733 BT734 BT735 CS001 CS101 CS201 CS202 CS205 CS206 CS301 CS302 CS304 CS310 CS311 CS312 CS314 CS315 CS401 CS402 CS403 CS405 CS407 CS408 CS409 CS410 CS411 CS431 CS432 CS435 CS501 CS502 CS504 CS506 CS507 CS508 CS510 CS601 CS602 CS604 CS605 CS606 CS607 CS609 CS610 CS611 CS614 CS615 CS620 CS701 CS702 CS703 CS704 CS706 CS707 CS708 CS709 CS710 CS711 CS712 CS713 CS716 CS718 CS721 CS723 CS724 CS726 ECO302 ECO303 ECO401 ECO402 ECO403 ECO404 ECO406 ECO501 ECO601 ECO602 ECO603 ECO704 EDU101 EDU201 EDU301 EDU303 EDU304 EDU305 EDU401 EDU402 EDU403 EDU404 EDU405 EDU410 EDU411 EDU430 EDU431 EDU501 EDU505 EDU510 EDU512 EDU515 EDU516 EDU601 EDU602 EDU603 EDU604 EDU654 EDU702 EDU705 ENG001 ENG101 ENG201 ENG301 ENG401 ENG504 ENG506 ENG507 ENG508 ENG509 ENG510 ENG511 ENG512 ETH201 ETH202 FIN611 FIN621 FIN622 FIN623 FIN624 FIN625 FIN630 FIN704 FIN711 FIN722 FIN723 FIN725 FIN730 GEN731 GSC101 GSC201 HRM611 HRM613 HRM617 HRM623 HRM624 HRM625 HRM626 HRM627 HRM628 HRM713 HRM724 HRM727 ISL201 IT430 MCD401 MCD402 MCD403 MCD501 MCD502 MCD504 MCM101 MCM301 MCM304 MCM310 MCM311 MCM401 MCM404 MCM411 MCM431 MCM501 MCM511 MCM514 MCM515 MCM516 MCM517 MCM520 MCM531 MCM532 MCM601 MCM604 MCM610 MGMT510 MGMT611 MGMT614 MGMT615 MGMT617 MGMT622 MGMT623 MGMT625 MGMT627 MGMT628 MGMT629 MGMT630 MGMT631 MGMT723 MGMT725 MGMT727 MGMT728 MGMT729 MGMT730 MGT101 MGT111 MGT201 MGT211 MGT301 MGT401 MGT402 MGT404 MGT411 MGT501 MGT502 MGT503 MGT504 MGT510 MGT513 MGT520 MGT522 MGT601 MGT602 MGT603 MGT604 MGT610 MGT611 MGT612 MGT613 MGT621 MGT703 MGT704 MGT705 MGT711 MGT713 MGT722 MKT501 MKT529 MKT530 MKT610 MKT611 MKT621 MKT624 MKT625 MKT627 MKT630 MKT703 MKT711 MKT721 MKT724 MKT725 MKT730 MKT731 MTH001 MTH100 MTH101 MTH201 MTH202 MTH301 MTH302 MTH303 MTH401 MTH403 MTH501 MTH601 MTH603 MTH631 MTH632 MTH633 MTH634 MTH641 MTH701 MTH706 MTH721 PAK301 PAK302 PHY101 PHY301 PSC201 PSC401 PSY101 PSY401 PSY402 PSY403 PSY404 PSY405 PSY406 PSY407 PSY408 PSY409 PSY502 PSY504 PSY505 PSY510 PSY511 PSY512 PSY513 PSY514 PSY515 PSY610 PSY631 PSY632 SOC101 SOC401 SPT730 STA301 STA304 STA404 STA406 STA408 STA621 STA630 STA730 STAT730 URD101 ZOO502 ZOO731