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Foundations in Microbiology Chapter 10 PowerPoint to accompany Fifth Edition Talaro Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Genetic engineering

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  • Foundations in MicrobiologyChapter10PowerPoint to accompanyFifth EditionTalaroCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

  • Genetic Engineering: A Revolution in Molecular BiologyChapter 10

  • Genetic engineeringdirect, deliberate modification of an organisms genomebioengineering

    Biotechnology use of an organisms biochemical and metabolic pathways for industrial production

  • I. Tools & Techniques of genetic engineeringenzymes for dicing, splicing, & reversing nucleic acidsanalysis of DNA

  • Enzymes for dicing, splicing, & reversing nucleic acidsrestriction endonucleases recognize specific sequences of DNA & break phosphodiester bondsligase rejoins phosphate-sugar bonds cut by endonucleasesreverse transcriptase makes a DNA copy of RNA - cDNA

  • Analysis of DNAgel electrophoresis- separates DNA fragments based on sizenucleic acid hybridization & probes probes base pair with complementary sequences; used to detect specific sequencesDNA Sequencing reading the sequence of nucleotides in a stretch of DNAPolymerase Chain Reaction way to amplify DNA

  • Gel electrophoresis

  • Southern blot hydridization

  • In situ hybridization

  • Sanger DNA sequence technique

  • Polymerase chain reaction (PCR)

  • II. Methods in Recombinant DNA Technologyconcerned with transferring DNA from one organism to another

    Cloning vectors & hostsConstruction of a recombinant plasmid

  • Characteristics of cloning vectorsmust be capable of carrying a significant piece of donor DNAmust be readily accepted by the cloning host

    plasmids small, well characterized, easy to manipulate & can be transferred into appropriate host cells through transformation bacteriophages have the natural ability to inject their DNA into bacterial hosts through transduction

  • Vector considerationsorigin of replicationsize of donated DNA vector will acceptgene which confers drug resistance to their cloning host

  • pBR322

  • Characteristics of cloning hostsrapid overturn, fast growth ratecan be grown in large quantities using ordinary culture methodsnonpathogenicgenome that is well delineated capable of accepting plasmid or bacteriophage vectorsmaintains foreign genes through multiple generationswill secrete a high yield of proteins from expressed foreign genes

  • III. Biochemical Products of Recombinant DNA Technologyenables large scale manufacturing of life-saving hormones, enzymes, vaccinesinsulin for diabeteshuman growth hormone for dwarfismerythropoietin for anemiaFactor VIII for hemophiliaHBV vaccine

  • IV. Genetically Modified Organisms (GMO)Recombinant microbes Pseudomonas syringae prevents ice crystalsBacillus thuringienisis encodes an insecticideTransgenic plantsRice that makes beta-caroteneTobacco resistant to herbicidesPeas resistant to weevilsTransgenic animalsMouse models for CF, Alzheimers, sickle cell anemiaSheep or goats that make medicine in their milk semen

  • Bioengineering of plants

  • Transgenic mice

  • V. Genetic TreatmentsGene therapyAntisense DNATriplex DNA

  • Gene therapycorrect faulty gene in human suffering from diseaseex vivo normal gene is is added to tissues taken from the body, then transfected cells are reintroduced into the bodyin vivo naked DNA or viral vector is directly introduced into patients tissueMost trials target cancer, single gene defects & infectionsMost gene deliveries are carried out by viral vectors

  • Gene therapy

  • Antisense DNA: targeting mRNAAntisense a nucleic acid strand with a base sequence that is complementary to the translatable strandAntisense DNA gets into the nucleus and binds to mRNA, blocking the expression of an unwanted proteincancersAlzheimers diseaseautoimmune diseases

  • Triplex DNAA triple helix formed when a third strand of DNA inserts into the major groove, making it inaccessible to normal transcriptionoligonucleotides have been synthesized to form triplex DNA oncogenes virusesreceptor for IL-2

  • Antisense DNA & triplex DNA

  • VI. Genome AnalysisGene MappingDNA Fingerprinting Microarray analysis

  • Gene Mappingdetermining the location of specific genes on the chromosomesHuman Genome Project to determine the nucleotide sequence of the >30,000 genes in the genome & the importance of these sequences & how they relate to human disease

  • Map of chromosome 16

  • DNA Fingerprinting Every individual has a unique sequence of DNAUsed to:identify hereditary relationshipsstudy inheritance of patterns of diseasesstudy human evolutionidentify criminals or victims of disaster

  • DNA fingerprints

  • Pedigree analysis

  • Microarray analysisMethod of determining which genes are actively transcribed in a cell under various conditionshealth vs diseasegrowth vs differentiationcould improve accuracy of diagnosis and specificity of treatment

  • Microarray