recombinant DNA

  1.  the process of altering genetic material by the purposeful manipulation of DNA
    Genetic engineering 
  2. Restriction enzymes (REs) cut DNA at specific 4 – 8 base pair(bp) sequences called these which are usually palindromes
     restriction sites
  3. The DNA fragments produced from restriction enzymes are called
    restriction fragments
  4. REs exist naturally in bacteria,protecting them from what?
    invading viral DNA
  5. these are the type of restriction enzymes normally used in genetic engineering
    Restriction endonucleases
  6. what do bacteria do to their DNA, protecting it from REs?
    methylate it
  7. Production of sticky ends by REs allows what to happen?
    the insertion of a foreign piece of DNA, as long as it has the same(complementary) sticky ends
  8.  the production of multiple copies of a gene of interest
    Gene cloning 
  9. this is an agent used to transfer DNA
    A vector
  10. what are the 2 most commonly used vectors?
    • bacterial plasmids
    • viruses
  11. how is cDNA made? 
    • by isolating mRNA molecules from the cell
    • using reverse transcriptase to make a complementary DNA molecule (cDNA)
  12. what is the advantage of using cDNA for cloning?
    it lacks introns, making it easier for a bacterium to express the correct protein 
  13. discuss gene cloning. 
    • isolate DNA from 2 sources
    • cut both with same RE
    • mix the DNA; they will join together because of the base pairing 
    • add DNA ligase to bond covalently
    • put plasmid into bacteria via transformation
    • identify cells with the recombinant DNA by their ability to grow in the presence of ampicillin but tetracyline
    • clone selected bacteria
  14. this is a large plasmid that contains just the genes necessary for replication (can carry an insert of 100-300 kb)
    The BAC (bacterial artificial chromosome)
  15. To find the clone, you must create and screen this
    a genomic “library” of clones
  16. You screen the library by creating a radioactive RNA probe that will bind to the gene you are interested in- what is this called?
    nucleic acid hybridization
  17. discuss how one would screen a genomic library.
    • transfer cells to filter 
    • treat cells on the filter to denature them
    • add radioactive probe to filter
    • expose the filter paper to photographic film
    • the colonies containing your gene will expose the film because they are radioactive
    • go back to the original plate, scoop out the colony you are interested in, and grow it in mass quantity 
  18. screening library using multiwell plates
    • The contents of each multiwell plate is transferred to a nylon membrane
    • it is then incubated in a plastic bag containing the radioactive probe
    • nylon membrane is then allowed to expose x-ray film
  19. this contains a highly active bacterial promoter just upstream from the restriction site where you insert the eukaryotic gene
    expression vector
Card Set
recombinant DNA
recombinant DNA