Chapter 9

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Chapter 9
2010-06-03 20:40:29
Biotechnology Recombinant DNA

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  1. Biotechnology
    • the use of microorganisms, cells ro cell compoennts to make a product
    • ex. foods, antibiotics, enzymes, proteins, vitamins
  2. Genetic engineering and Recombinant DNA technolgoy
    insertion of genes in an organism to produce desired protesin/enzymes or creat new cells that produce chemicals that an organism doesn't naturally make.
  3. Selection
    • the culturing of a naturally-occurring microbe that produces a desired product.
    • Two types:
    • 1. Artificial selection
    • 2.Natural selection
  4. Natural selection
    survival of the fittest in biotechn.
  5. Artificial selection
    selectiion by humans of organisms with desirable characteristics.
  6. Mutation
    • mutagens cause mutations that might result in a microbe with a desirable trait.
    • Two types:
    • 1. Site-directed mutagenesis
    • 2. Random selection
  7. Mutation - Site-directed mutagenesis
    changing a specific gene in DNA to change a protein
  8. Mutation - random selection
    select and culture microbe with the desired mutation.
  9. Restriction enzymes
    • cut specific sequences of DNA
    • Naturally occur in some species of bacteria. ex. to protect against incoming DNA to cut it up before it cuases any damage.
    • Methylated cytosins - tells restriction enzymes not to cut its own DNA
  10. different Restriction enzymes
    • cuts are staggered, producing "skicky ends" - become single strands
    • if two fragments of DNA from different sources have been cut with the same restriction enzyme, the two fragments will have complimentroy sticky ends and can recombine.
    • DNA ligase will then covalently link the sugar-phosphate backbones, producing a recombinant DNA molecule.
  11. Vectors
    • Transport DNA into the desired cell
    • ex. Plasmids, transposons, and viruses
  12. Importanc characterists of Vectors
    • Must be self replicating
    • Small
    • Should resist destruction by the recipient cell
    • a. circular vectors are highly resilient
    • b. Viral DNA more likely to remain intact.
    • Should carry a marker gene, which makes it easy to retrieve clones containing the vector.
  13. Shuttle vectors
    are plasmids capable of existing in several different species, and are used to move genes from one species to another.
  14. Gene therapy and vectors
    Vectors used to insert functional genes into human cells that have defective genes.
  15. Polymerase Chain Reaction (PCR)
    used to make multiple copies of a piece of DNA.
  16. PCR is used to:
    • Clone DNA for recombination
    • Amplify DNA to detectable levles
    • Sequence DNA - to determain order of letters
    • Diangose genetic disease
    • Detect pahtognes
  17. Steps of PCR:
    • 1.Original piece of DNA placed in a test tube with DNA primers, Individual DNA necluotides, DNA polymerease. Than test tube placed in a thermalcycler.
    • 2.Incubated at 94 C for 1 min to seperate the strands of the original piece of DNA
    • 3. Temp lowered to 60 C for 1 min. - DNA primers attach to the individual DNA strands
    • 4. Temp increased to 72 C for 1 min - DNA polymerease synthesized a complimentory strand of DNA
    • 5. Cycle is repeated many times, resulting in an exponential increase in the number of DNA pieces, all are identical to original DNA
    • 6. DNA polymerase taken from the thermophilic bacterium "Thermus aquaticus" becuase of high temps.
  18. Ways a recombinant DNA can be inserted into a cell
    • Transformation
    • Electroporation
    • Protoplast Fusion
    • Gene gun
    • Microinjeciton
  19. Transformation
    cell treated with chemicals to make them "competent" or able to take up external DNA. Ex. CaCl2, than heat shocked
  20. Electroporation
    • applied an electrical current that forms small pores in the plasma membrane so the DNA can pass through.
    • To take up DNA better by those cell that have cell walls they need to be converted to protoplasts. (G + doesn't have cell walls, so thye take up DNA beter)
  21. Protoplasts fusion
    Protoplasts put into a solution with added polyethylene glycol that increases the rate of fusion of two cells so that the 2 chromosomes can undergo natural recombination and become one cell.
  22. Gene gun
    pariticles of gold are coated with DNA and shot out of a gne gun witha burst of helium so that it penetrates plant cell walls - doen to eukaryotes - they can survive better.
  23. Microinjection
    a glass micropipette is used to inject DNA into a cell - humans
  24. Gene library
    are made up of pieces of an entire genome stored in bacterial plasmids or in phages.
  25. Synthetic DNA
    made by a DNA synthesis machine - only short pieces
  26. Cloning the genes of Eukaryotes
    • Complementary DNA (cDNA) is made from an mRNA template by the enzyme reverse transcriptase.
    • The result is a DNA molecules without introns.
    • This is used to obtain eukaryotic genes that can then be inserted into a prokaryotes.
  27. Selecting a clone
    • from a gene library that contains your gene of interests
    • two ways:
    • 1. Blue-white screening
    • 2. Colony hybridization
  28. Blue-white screening
    • selects for clones that have any recombinant DNA
    • Process:
  29. Colony hybridization
    • is specific: identifies the clone with your gene of interest.
    • DNA probes are short sequences of single-stranded DNA that are compoimentary to the desired gene.
    • a. DNA probes are synthesized in the labe
    • b. Some DNA probes contain fluorescent dye
    • c. Some DNA probes contain radiocative phosphorous
    • DNA probes will bind with the gene of interest and serve as indicators