Genetic Engineering

Paperback | December 31, 2010

bySmita Rastogi, Neelam Pathak

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Beginning with the basic concept of gene cloning, topics such as restriction endonucleases, chemical synthesis of oligonucleotides, cDNA synthesis, plasmids, and polymerase chain reaction have been discussed in detail in this book. The book also contains separate chapters on molecular marketsand public concerns. All chapters begin with the chapter objectives and provide a exhausitive recap of the important concepts at the end of the chapter followed by review questions and numerical exercises. The language is extremely lucid and pertinent and interesting facts and anecdotes have also been included in each chapter which should surely hold the interest of readers.

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Beginning with the basic concept of gene cloning, topics such as restriction endonucleases, chemical synthesis of oligonucleotides, cDNA synthesis, plasmids, and polymerase chain reaction have been discussed in detail in this book. The book also contains separate chapters on molecular marketsand public concerns. All chapters begin with...

Dr Smita Ratogi and Dr Neelam Pathak are faculty members in the department of Biotechnology, Integral University, Lucknow. They have been teaching 'Genetic Engineering' and 'Molecular Biology' to postgraduate and undergraduate students. Both of them are PhDs in Biochemistry from Lucknow University. They have several articles and publi...
Format:PaperbackDimensions:652 pages, 11.25 × 8.75 × 0.68 inPublished:December 31, 2010Publisher:Oxford University PressLanguage:English

The following ISBNs are associated with this title:

ISBN - 10:0195696573

ISBN - 13:9780195696578


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Table of Contents

PrefaceAbbreviations1. Basic concept of gene cloningDefinition of genetic engineeringOutline of the process2. Enzymes used in genetic engineeringRestriction endonucleaseDNA polymerase:DNA Pol I, Klenow fragmentT4 DNA polymerase (Filling in and Trimming back)Thermostable DNA polymerasesTerminal deoxynucleotidyl transferaseRNA PolymeraseReverse transcriptaseAlkaline phosphatasePolynucleotide kinaseDNA ligase:E. coli DNA ligaseT4 DNA ligaseDeoxyribonucleaseRibonuclease3. Restriction endonucleasesDefinitionDiscoveryClassification: Type I, II and IIINomenclatureRecognition sites:Palindrome, Two-fold axis of symmetrySpecificity and mode of action Cognate methylasesRestriction enzymes in genetic engineering:Sticky end cutters (definition, examples and advantages)Blunt end cutters (definition, examples and advantages)Rare base cutters (definition, examples and advantages)Isoschizomers (definition, examples and advantages)Heterohypekomers (definition, examples and advantages)List of restriction enzymes (nomenclature; recognition; cleavage sites)Unit of restriction enzyme activityRestriction digestion: partial and complete digestionStar activityRestriction digestion vs mechanical shearing method of generation of DNA fragmentsApplication of restriction enzymes in restriction mapping4. Chemical synthesis of oligonucleotidesWhy and when opt for chemical synthesis?Methods of chemical synthesis (details of the process; efficiency; advantages/ disadvantages):Phosphodiester methodPhosphotriester methodPhosphoramidite methodPhotolithographic method5. cDNA synthesisIsolation of RNA and purification of mRNAcDNA vs genomic DNARole of various activities of reverse transcriptaseVarious methods for first and second strand DNA synthesis (use of oligo-dT primers, random primers in first strand synthesis; use of replacement method, asymmetrically-tailed plasmid, oligonucleotide primers, random primers in second strand synthesis)RT-PCR6. Subfractionation of DNA fragmentsConventional agarose gel electrophoresis (principle; use of LMP; elution from gel)Polyacrylamide gel electrophoresis (principle)Pulsed Field Gel Electrophoresis (PFGE) (principle; variants)Density gradient centrifugationGel filtration chromatography7. Polymerase Chain ReactionWhy and when use PCR?Principle of PCRVariants of PCR:DD-RTPCRDegenerate PCRAsymmetric PCRInverse PCRAnchored PCRReal time PCRScorpion PCR RT-PCRError prone PCR Applications of PCR [Isolation of gene; PCR product cloning (TA cloning, Topo cloning); PCR based mutagenesis; cDNA synthesis; Rapid Amplification of cDNA Ends (RACE); PCR based molecular markers]8. PlasmidsBiology of plasmids:DefinitionWhy is plasmid not considered genome?Plasmid size rangePlasmid shapes: covalently closed circular, linear plasmidsPlasmid classification on basis of phenotypic traits: Cryptic plasmid, Fertility (F)/ Conjugative plasmid Resistance (R) plasmid (mode of action of resistance genes) Bacteriocinogenic plasmid (mode of action of toxins), Degradative plasmid (mode of action of degrading genes), Virulence plasmidMobilizable plasmidPlasmid host rangeRelaxed and stringent control of plasmid copy numberGenome map of Col E1 plasmidPlasmid as cloning vector (recombinant plasmids):Advantages of using plasmid vectorsExamples of plasmids: pBR322 (vector map; strategy of construction), pUC (vector map; strategy of construction)9. Lambda phagesBiology of y phage:Genome map of y phageFunctions of various gene productsLysogeny and lytic cycleImportance of cos sitesy phage as cloning vector:Advantages of using phage lambda vectorInsertional and replacement vectors (definition; advantage/ disadvantage; examples; vector maps of important vectors)Importance of some terms associated with cloning in y vector:Insert capacity and packaging constraint, Temperature sensitive repressor, hfl A mutant host Amber mutations in lytic genes, rec A- host, Spi phenotype10. M13 phagesBiology of M13 phage:Genome map of M13 phageFunctions of various gene productsPhage assemblyStructural and functional differences from y phageM13 phage as cloning vector:M13 series of vectors (important examples, geneology, advantages, sister vectors, hosts used)11. Other vectorsChimeric vectors:Cosmid (definition; advantages; single cos and dual cos cosmids; examples)Phagemid (definition, advantages, examples, hosts and helper phages used)Phasmid (definition, advantages, examples)Fosmid (definition, advantages, examples)Tumor inducing plasmid (pTi) from Agrobacterium:Introduction to AgrobacteriumApplication in plant transformationGenome map of pTiFunctions of various gene productspTi based vectors: Binary and cointegrate vector system (definition; examples; strategy of construction; advantages / disadvantages)Viral vectors:Advantages of using viral system as cloning vectorPlant viral vectorsAnimal viral vectorArtificial chromosomes:Bacterial artificial chromosome (BAC)P1-derived artificial chromosome (PAC)Bacillus subtilis, Streptomyces, Pseudomonas based vectors (advantages of cloning in non - E. coli systems)Shuttle vectorExpression vector (examples with vector maps; importance of tissue specific, wound inducible, strong, regulatable, T7 and T3 promoters, Details of CaMV 35S promoter)Fusion vector (Translational and transcriptional fusion vectors; advantages of fusion proteins)Specialist vector (for protein secretion, protein solubilization, purification tags, surface display, production of RNA probes)Advanced gene tagging/ trapping vectors (Gene trap vector, Plasmid rescue vector, Enhancer trap vector, Activation tagging)12. Yeast cloning vectorsWhy is a yeast system required?Types of yeast vectors:2u plasmidYeast integrative plasmid (YIp)Yeast episomal plasmid (YEp)Yeast replicative plasmid (YRp)Yeast centromeric plasmid (YCp)Yeast linear replicative plasmid (Ylp)Yeast artificial chromosome (YAC)Other fungal systems13. Joining of DNA fragmentsDNA ligaseHomopolymer tailing (definition; advantages/ disadvantages)Linkers (definition; advantages/ disadvantages)Adaptors (definition; advantages)14. DNA delivery methodsMethods for DNA delivery to bacterial system:ConjugationMobilization and Triparental matingChemical inductionPreparation of competent cells and transformation by heat-shock or freeze-thaw methodIn vitro packaging and natural infectionTransductionTransfectionUltrasonicationVirus/ phage mediated genetic transformationDNA delivery to prokaryotic non - E. coli systemsDNA delivery to yeast and other fungal systemsMicroprojectile bombardmentElectroporationMicroinjectionDNA delivery into plants/ animalsAgrobacterium mediated genetic transformation and integration of exogenous DNA into plant genomeVirus mediated genetic transformationMicroprojectile bombardmentElectroporationMicroinjectionLiposome packagingProtoplast fusionOther uncommon methods: use of silicon carbide whiskers, ultrasonicationDNA integration into bacterial genomeDNA delivery to prokaryotic non - E. coli systemsDNA delivery to yeast and other fungal systems15. Cloning strategies using different vector systemsLibrary:Genomic library (construction; calculation for probability to clone the desired DNA fragment)cDNA library (construction; effect of mRNA abundance)Genomic library vs cDNA library (advantages/ disadvantages)PCR product cloningTA cloningTopo cloningRT-PCR (an alternative to cDNA cloning)Strategy for cloning in plasmid vectorStrategy for cloning in y insertion vectorStrategy for cloning in y replacement vectorStrategy for cloning in single cos cosmid vectorStrategy for cloning in dual cos cosmid vectorStrategy for cloning in yeast artificial chromosomeUse of linkers, adapters, homopolymer tailing in cloning16. Selection/ Screening of recombinants/ transformantsPreparation of probe DNA (radioactive and non-radioactive labeling by methods as nick translation; end filling; random primer methods)Overview of techniques for recombinant selection and screeningFunctional (genetic) complementation (blue-white screening; red-white screening; importance of lac Z' and sup 4)Nutritional complementation (auxotrophic mutants)Gain of functionColony hybridizationPlaque hybridizationSouthern blotting and hybridizationDNA and RNA dot blotZoo blotPlus-Minus screeningNorthern blotting, Reverse Northern blottingImmunological screeningWestern blottingSouth-Western blottingNorth-Western blottingHybrid arrest translation (HART) and Hybrid release translation(HAT)DNA chip17. Other techniques for gene manipulationMutagenesis and recombinationSite directed mutagenesisTransposon mutagenesisChemical mutagenesisInsertional mutagenesisPCR based mutagenesisSite-specific recombinationPost Transcriptional Gene SilencingAntisense RNA technologyRNA interferenceCosuppression18. Techniques used in Genomics and ProteomicsGenomics:Rapid DNA and RNA sequencing techniques: Sanger method, Maxam and Gilbert procedureAutomated DNA sequencingPyrosequencingHigh throughput Sequencing: Shot gun cloning, Chromosome walking, Clone contig assembly, AnnotationMicroarrayProteomics:Protein sequencing: N and C terminal sequencing, Edman degradation, Dansyl chloride2-D electrophoresisMulti-D liquid chromatographyMass spectrometryMALDI-TOFYeast two-hybrid systemDNase I foot printingProtein Microarray19. Applications of genetic engineeringCloning in plant cells:Developing insect resistance, disease-resistance, herbicide resistant, salt and submergence stress tolerant plantsQuality improvementEdible vaccinesApplications in biodiversity conservationCloning in mammalian cells:Therapeutic cloningReproductive cloningOrigins of organismal cloning in developmental biology-search on frogsNuclear transfer proceduresCloning of sheep (Dolly) and other mammalsGene knockout technologyTransgenicsGene therapyStem cell therapy20. Public concernsSafety guidelines of recombinant DNA research (Containment facilities and disposal of radioactive material; Protection from exposure of ultraviolet light, ethidium bromide etc.)Ethical issues and prospects for human cloningHealth and environmental concerns related to genetically modified organisms (GMOs)