1 / 20

Chapter 9

Biotechnology and Recombinant DNA. Chapter 9. Biotechnology and Recombinant DNA. SLOs. Compare and contrast biotechnology, recombinant DNA technology, and genetic engineering. Identify the roles of a clone and a vector in making recombined DNA. Compare selection and mutation.

karlyn
Télécharger la présentation

Chapter 9

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Biotechnology and Recombinant DNA Chapter 9 Biotechnology and Recombinant DNA

  2. SLOs Compare and contrast biotechnology, recombinant DNA technology, and genetic engineering. Identify the roles of a clone and a vector in making recombined DNA. Compare selection and mutation. Define restriction enzymes, and outline their use to make recombinant DNA. List some properties of vectors and describe their use. Outline the steps in PCR and provide an examples of its use. Describe various different ways of getting DNA into a cell. Differentiate cDNA from synthetic DNA. Explain how each of the following are used to locate a clone: antibiotic-resistance genes, DNA probes, gene products. Outline advantages of engineering with either E. coli, Saccharomycescerevisiae, mammalian cells, or plant cells. List some advantages of, and problems associated with, the use of genetic modification techniques.

  3. Terminology and Definitions • Biotechnology: Manipulation (as through genetic engineering) of living organisms or their components to produce useful commercial products • Recombinant DNA (rDNA) technology: Insertion or modification of genes to produce desired proteins • Clones and cloning: • Cloning vectors (plasmids and viruses)  recombinant vectors • Cell clones • “to clone a gene” - question: Interest in DNA or in gene product?

  4. Biotechnology Tools • Natural vs. artificial selection • Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait. Now: Site-directed mutagenesis • Restriction Enzymes (RE): Molecular scissors • Cut specific sequences of DNA • Destroy bacteriophage DNA in bacterial cells • Methylases protect own DNA by methylatingcytosines • Bunt ends vs. sticky ends Review Microbiology Animations with Quizzes in Mastering Microbiology

  5. Site of cleavage Restriction Enzymes (= Restriction Endonucleases) EcoRI Recognition sequence is always a palindrome Fig 8-25 Review Table 9.1

  6. Origin and Naming of Restriction Enzymes

  7. Role of Restriction Enzymes in Making Recombinant DNA Molecules Fig 9.2

  8. Cloning Vectors • Must be self-replicating in large quantities • are recombinant DNA molecules. • introduce foreign DNA into host cells Plasmids and viruses are commonly used vectors. Shuttle vectors can exist in several different species. Fig 9.3

  9. Polymerase Chain Reaction (PCR) • Makes multiple copies of a piece of DNA enzymatically • Used to • Clone DNA for recombination • Amplify DNA to detectable levels • Sequence DNA • Diagnose genetic disease • Detect pathogens For details see Lab Fig 9.4 Review Microbiology Animations with Quizzes in Mastering Microbiology

  10. Inserting Foreign DNA into Cells • DNA can be inserted into a cell by • Transformation • Electroporation • Protoplast fusion • Microinjection Fig 9.7

  11. BlueandWhiteScreening Methodfor Selecting a Clone (or Recombinant DNA Molecule) Direct selection of engineered vector via antibiotic-resistance markers (ampR) on plasmid vectors. Vector also contains-galactosidase gene for blue-white screening Desired gene is inserted into the -galactosidase gene site  gene inactivated Possible outcomes: • Bacterial clones contain recombinant vector  resistant to Ampicillin and unable to hydrolyze X-gal (white colonies). • Bacterial clones contain vector without the new gene  blue colonies. • Bacteria lack vector  will not grow.

  12. Possible Method to detect recombinant bacteria: Blue–White Screening Fig 9.11

  13. Which type of colonies do you want? White Blue I don’t want any

  14. Making a Gene Product E. coli: prokaryotic workhorse of biotechnology (easily grown and its genomics well understood). Need to eliminate endotoxin from products Cells must be lysed to get product Yeast:Saccharomyces cerevisiae is eukaryotic workhorse of biotechnology. Continuous secretion of gene product. Mammalian cells: May express eukaryotic genes easily. Harder to grow. Plant cells: Easy to grow. May express eukaryotic genes easily.

  15. Some Biotechnology (rDNA) Applications Diagnostics: PCR and DNA probes can be used to quickly identify a pathogen in body tissue or food. (Forensic microbiology) Therapeutic Applications: • Insulin production • Subunit vaccines • DNA vaccines • Gene therapy to replace defective or missing genes • Pharmaceutical applications • Hormone and Antibiotics production

  16. Transformation Cloning genes for Insulin production

  17. Safety Issues and Ethics of Using rDNA Strict safety standards avoid accidental release of genetically modified microorganisms. Some microbes used in cloning have been altered so that they cannot survive outside the laboratory. Microorganisms intended for use in the environment may be modified to contain suicide genes  organisms do not persist in the environment. Safety and ethical concerns beyond microbiology: Who will have access to an individual's genetic information? Are genetically modified crops safe for release to environment?

  18. A Typical Genetic Modification Procedure Foundation Figure Fig 9.1 Fig 9.1

  19. A Typical Genetic Modification Procedure Fig 9.1 cont.

  20. The End

More Related