Chapter 9 Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA
Q&A Interferons are species specific, so that interferons to be used in humans must be produced in human cells. Can you think of a way to increase the supply of interferons so that they can be used to treat diseases?
Objectives 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, 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 Genetic engineering: Techniques used to cut up and join together genetic material (from different species) and to introduce the result into an organism in order to change one or more of its characteristics.
Biotechnology Tools Selection and Mutation Artificial selection: Culture a naturally occurring microbe that produces desired product Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait. Sitedirected mutagenesis: Restriction Enzymes (RE): Molecular scissors Cut specific sequences of DNA Destroy bacteriophage DNA in bacterial cells Methylases protect own DNA by methylating cytosines ANIMATION: Recombinant DNA Technology
Site of cleavage Restriction Enzymes (= Restriction Endonucleases) Fig 8-25 Recognition sequence is always a palindrome
Origin and Naming of Restriction Enzymes
Role of Restriction Enzyme in Making Recombinant DNA Molecules Figure of the week: Fig 9.2
Cloning Vectors are recombinant DNA molecules. introduce foreign DNA into host cells are self-replicating in large quantities Plasmids and viruses are commonly used vectors. Shuttle vectors can exist in several different species.
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 PCR Animation ANIMATION PCR: Overview ANIMATION PCR: Components
PCR Figure of the week: Fig 9.4
Inserting Foreign DNA into Cells DNA can be inserted into a cell by Transformation Electroporation Protoplast fusion Microinjection Fig 9.5
Obtaining DNA Genomic libraries: genes stored in plasmids or phages The stored genes can be natural copies of genes. Exons and introns in Eukaryotes! made from mrna by reverse transcriptase (cdna). synthetic DNA made by a DNA synthesis machine.
Obtaining DNA Complementary DNA (cdna) is made from mrna by reverse transcriptase Fig 9.9
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 Possible outcomes: 1. Bacterial clones contain recombinant vector resistant to ampicillin and unable to hydrolyze X-gal (white colonies). 2. Bacterial clones contain vector without the new gene blue colonies. 3. Bacteria lack vector will not grow.
Fig 9.11 Possible Method to detect recombinant bacteria: Blue White Screening
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.
Some Biotechnology Applications Diagnostics: PCR and DNA probes can be used to quickly identify a pathogen in body tissue or food. (Forensic microbiology) Gene therapy to replace defective or missing genes Pharmaceutical applications Hormone and Antibiotics production Vaccines (subunit vaccines, DNA vaccines, nonpathogenic viruses carrying genes for pathogen's antigens as vaccines)
Transformation Cloning genes
Forensic Microbiology PCR Primer for a specific organism will allow for detection if that organism is present Real-time PCR: Newly made DNA tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle Reverse-transcription (RT- PCR): Reverse transcriptase makes DNA from viral RNA or mrna RT-PCR with a norovirus primer Clinical Focus, p. 266
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 Fig 9.1
A Typical Genetic Modification Procedure Fig 9.1