Chapter 9 Biotechnology and DNA Technology
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 REs, 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. 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, Saccharomyces cerevisiae, mammalian cells, or plant cells. List some advantages of, and problems associated with, the use of genetic modification techniques.
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 Overview of recombinant DNA Procedures: Cell clones to clone a gene - question: Interest in DNA or in gene product? vector
Biotechnology Toolkit 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 methylating cytosines Bunt ends vs. sticky ends
Restriction Enzymes = REs (= Restriction Endonucleases) Site of cleavage EcoRI Recognition sequence Fig 8-25 is always a palindrome Review Table 9.1
Origin and Naming of Restriction Enzymes
Role of Restriction Enzymes in Making Recombinant DNA Molecules Fig 9.2
Vectors Also known as cloning vectors. Must be Small and easy to manipulate. & serve as vectors. self-replicating large quantities When they carry insert : = Recombinant DNA molecules Introduce foreign DNA (desired gene) into host cells Shuttle vectors can exist in several different species.
... One of most commonly used vectors:
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 Review Microbiology Animations with Quizzes in Mastering Microbiology Review Fig 9.4
Techniques of Genetic Modification Inserting Foreign DNA into Cells DNA can be inserted into a cell by Transformation Protoplast fusion Electroporation Microinjection Fig 9.5
Blue and White Screening Method for Selecting a Clone (or Recombinant DNA Molecule) Direct selection of engineered vector via antibioticresistance markers (ampr) on plasmid vectors. Vector also contains -galactosidase gene for bluewhite screening Desired gene is inserted into the -galactosidase gene site gene inactivated
Three possible outcomes: 1. Bacteria lack vector 2. Bacterial clones contain vector without the new gene colony type? 3. Bacterial clones contain recombinant vector resistant to Ampicillin and unable to hydrolyze X-gal colony type? 1) Plasmid cloning Fig 9.11
2) Selecting Recombinant Bacteria
Which type of colonies do you want? a)white b)blue c)i don t want any
Making a Gene Product E. coli: prokaryotic workhorse of biotech. Easily grown and genomics well understood. Disadvantage: Cells must be lysed to get product release of Yeast: Saccharomyces cerevisiae is eukaryotic workhorse of biotechnology. Advantage: Continuous secretion of gene product. Mammalian cells: May express eukaryotic genes easily. Disadvantage: Harder to grow. Plant cells: Easy to grow. May express eukaryotic genes easily.
Some Applications of DNA Technology Forensic Microbiology & Diagnostics: PCR and DNA probes can be used to quickly identify a pathogen in body tissue or food. Therapeutic Applications: 1. Pharmaceutical applications, e.g.: Insulin production 2. Subunit vaccines 3. DNA vaccines 4. Gene therapy to replace defective or missing genes 5. Gene silencing See Table 9.2
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?
A Typical Genetic Modification Procedure Foundation Figure Fig 9.1