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Foundations in Microbiology. PowerPoint to accompany. Fifth Edition. Talaro. Chapter 10. Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Genetic Engineering: A Revolution in Molecular Biology. Chapter 10. Genetic engineering.

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Foundations in microbiology l.jpg

Foundations in Microbiology

PowerPoint to accompany

Fifth Edition

Talaro

Chapter

10

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.


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Genetic Engineering: A Revolution in Molecular Biology

Chapter 10


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Genetic engineering

  • direct, deliberate modification of an organism’s genome

  • bioengineering

  • Biotechnology – use of an organism’s biochemical and metabolic pathways for industrial production


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I. Tools & Techniques of genetic engineering

  • enzymes for dicing, splicing, & reversing nucleic acids

  • analysis of DNA


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Enzymes for dicing, splicing, & reversing nucleic acids

  • restriction endonucleases – recognize specific sequences of DNA & break phosphodiester bonds

  • ligase – rejoins phosphate-sugar bonds cut by endonucleases

  • reverse transcriptase – makes a DNA copy of RNA - cDNA


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Analysis of DNA

  • gel electrophoresis- separates DNA fragments based on size

  • nucleic acid hybridization & probes – probes base pair with complementary sequences; used to detect specific sequences

  • DNA Sequencing – reading the sequence of nucleotides in a stretch of DNA

  • Polymerase Chain Reaction – way to amplify DNA







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II. Methods in Recombinant DNA Technology

  • concerned with transferring DNA from one organism to another

  • Cloning vectors & hosts

  • Construction of a recombinant plasmid


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Characteristics of cloning vectors

  • must be capable of carrying a significant piece of donor DNA

  • must 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


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Vector considerations

  • origin of replication

  • size of donated DNA vector will accept

  • gene which confers drug resistance to their cloning host



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Characteristics of cloning hosts

  • rapid overturn, fast growth rate

  • can be grown in large quantities using ordinary culture methods

  • nonpathogenic

  • genome that is well delineated

  • capable of accepting plasmid or bacteriophage vectors

  • maintains foreign genes through multiple generations

  • will secrete a high yield of proteins from expressed foreign genes


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III. Biochemical Products of Recombinant DNA Technology

  • enables large scale manufacturing of life-saving hormones, enzymes, vaccines

    • insulin for diabetes

    • human growth hormone for dwarfism

    • erythropoietin for anemia

    • Factor VIII for hemophilia

    • HBV vaccine


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IV. Genetically Modified Organisms (GMO)

  • Recombinant microbes

    • Pseudomonas syringae – prevents ice crystals

    • Bacillus thuringienisis –encodes an insecticide

  • Transgenic plants

    • Rice that makes beta-carotene

    • Tobacco resistant to herbicides

    • Peas resistant to weevils

  • Transgenic animals

    • Mouse models for CF, Alzheimer’s, sickle cell anemia

    • Sheep or goats that make medicine in their milk semen




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V. Genetic Treatments

  • Gene therapy

  • Antisense DNA

  • Triplex DNA


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Gene therapy

  • correct faulty gene in human suffering from disease

    • ex vivo – normal gene is is added to tissues taken from the body, then transfected cells are reintroduced into the body

    • in vivo – naked DNA or viral vector is directly introduced into patient’s tissue

  • Most trials target cancer, single gene defects & infections

  • Most gene deliveries are carried out by viral vectors



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Antisense DNA: targeting mRNA

  • Antisense – a nucleic acid strand with a base sequence that is complementary to the translatable strand

  • Antisense DNA gets into the nucleus and binds to mRNA, blocking the expression of an unwanted protein

    • cancers

    • Alzheimer’s disease

    • autoimmune diseases


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Triplex DNA

  • A triple helix formed when a third strand of DNA inserts into the major groove, making it inaccessible to normal transcription

  • oligonucleotides have been synthesized to form triplex DNA

    • oncogenes

    • viruses

    • receptor for IL-2



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VI. Genome Analysis

  • Gene Mapping

  • DNA Fingerprinting

  • Microarray analysis


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Gene Mapping

  • determining the location of specific genes on the chromosomes

  • Human 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



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DNA Fingerprinting

  • Every individual has a unique sequence of DNA

  • Used to:

    • identify hereditary relationships

    • study inheritance of patterns of diseases

    • study human evolution

    • identify criminals or victims of disaster




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Microarray analysis

  • Method of determining which genes are actively transcribed in a cell under various conditions

    • health vs disease

    • growth vs differentiation

  • could improve accuracy of diagnosis and specificity of treatment



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