In the name of ALLAH, the most beneficient, the most merciful

Gene Manipulation and Genetic Engineering

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Course Code: BIO732

Instructor: Dr. Basharat Ali

Total Lectures: 230

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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

Course Codes

ACC311 ACC501 ACC707 BIF101 BIF401 BIF402 BIF501 BIF601 BIF602 BIF604 BIF731 BIF732 BIF733 BIO101 BIO102 BIO201 BIO202 BIO203 BIO204 BIO301 BIO302 BIO303 BIO401 BIO502 BIO503 BIO504P BIO505P BIO506P BIO731 BIO732 BIO733 BIO734 BIT701 BIT703 BIT710 BIT715 BNK601 BNK603 BNK604 BNK610 BNK611 BNK612 BNK613 BNK701 BNK703 BNK704 BNK725 BT101 BT102 BT201 BT301 BT302 BT401 BT402 BT403 BT404 BT405 BT406 BT501 BT503 BT504 BT505 BT511P BT512P BT513P BT513T BT514P BT514T BT601 BT603 BT604 BT605 BT611P BT613P BT614P BT731 BT732 BT733 BT734 BT735 CHE201 CHE301 CS001 CS101 CS201 CS202 CS204 CS205 CS206 CS301 CS302 CS304 CS310 CS311 CS312 CS314 CS315 CS401 CS402 CS403 CS405 CS406 CS407 CS408 CS409 CS410 CS411 CS431 CS432 CS435 CS442 CS501 CS502 CS504 CS506 CS507 CS508 CS510 CS511 CS601 CS602 CS603 CS604 CS605 CS606 CS607 CS608 CS609 CS610 CS611 CS612 CS614 CS615 CS620 CS642 CS701 CS702 CS703 CS704 CS706 CS707 CS708 CS709 CS710 CS711 CS712 CS713 CS716 CS718 CS721 CS723 CS724 CS725 CS726 ECE101 ECE201 ECE202 ECE203 ECE204 ECE301 ECE302 ECE402 ECO302 ECO303 ECO401 ECO402 ECO403 ECO404 ECO406 ECO501 ECO601 ECO602 ECO603 ECO604 ECO605 ECO606 ECO607 ECO608 ECO609 ECO612 ECO613 ECO614 ECO615 ECO616 ECO622 ECO704 EDU101 EDU201 EDU301 EDU302 EDU303 EDU304 EDU305 EDU306 EDU401 EDU402 EDU403 EDU404 EDU405 EDU406 EDU407 EDU410 EDU411 EDU430 EDU431 EDU433 EDU501 EDU505 EDU510 EDU512 EDU515 EDU516 EDU601 EDU602 EDU603 EDU604 EDU654 EDU702 EDU705 EDUA305 EDUA411 ENG001 ENG101 ENG201 ENG301 ENG401 ENG501 ENG502 ENG503 ENG504 ENG505 ENG506 ENG507 ENG508 ENG509 ENG510 ENG511 ENG512 ENG513 ENG514 ENG515 ENG516 ENG518 ENG519 ENG524 ENG527 ETH100 ETH201 ETH202 FIN611 FIN621 FIN622 FIN623 FIN624 FIN625 FIN630 FIN702 FIN703 FIN704 FIN711 FIN722 FIN723 FIN725 FIN730 GEN731 GEN732 GSC101 GSC201 HRM611 HRM613 HRM617 HRM623 HRM624 HRM625 HRM626 HRM627 HRM628 HRM630 HRM713 HRM724 HRM727 HRM730 HRM733 HRM737 ISL201 ISL202 IT430 MB501P MB502P MB503P MB504P MCD401 MCD402 MCD403 MCD404 MCD501 MCD502 MCD503 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 MGMT715 MGMT723 MGMT725 MGMT727 MGMT728 MGMT729 MGMT730 MGMT731 MGMT737 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 MGT701 MGT703 MGT704 MGT705 MGT711 MGT713 MGT714 MGT717 MGT722 MGT723 MGT725 MGTE630 MIC501P MKT501 MKT529 MKT530 MKT610 MKT611 MKT621 MKT624 MKT625 MKT626 MKT627 MKT630 MKT703 MKT711 MKT721 MKT724 MKT725 MKT726 MKT728 MKT730 MKT731 MKT740 MTH001 MTH100 MTH101 MTH201 MTH202 MTH301 MTH302 MTH303 MTH401 MTH403 MTH501 MTH601 MTH603 MTH631 MTH632 MTH633 MTH634 MTH641 MTH642 MTH701 MTH706 MTH7123 MTH721 PAD603 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 PSY516 PSY610 PSY611 PSY613 PSY631 PSY632 PSYP402 SOC101 SOC301 SOC302 SOC401 SOC402 SOC403 SOC404 SOC509 SOC601 SOC602 SOC603 SOC604 SOC605 SOC606 SOC607 SOC608 SOC609 SOC610 SOC611 SOC612 SOC613 SOC614 SOC615 SOC616 SOC617 SPT730 STA100 STA301 STA304 STA404 STA408 STA621 STA630 STA631 STA632 STA633 STA641 STA642 STA643 STA644 STA730 STAT406 STAT730 URD101 VU001F ZOO101 ZOO102 ZOO103 ZOO201 ZOO301 ZOO302 ZOO401 ZOO403 ZOO404 ZOO501 ZOO502 ZOO502P ZOO502T ZOO503 ZOO504 ZOO505 ZOO506 ZOO507 ZOO510 ZOO511T ZOO512T ZOO514P ZOO514T ZOO515P ZOO516P ZOO516T ZOO517P ZOO517T ZOO518P ZOO519P ZOO519T ZOO603P ZOO630 ZOO630P ZOO731