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Transcript

  • 1. Recombinant Tech II
  • 2. DNA MAPS
    • Genetic = recombination frequencies.
    • Cytological = banding pattern of chromosomes
    • Physical = Distances in bp. kb, megabase pairs.
  • 3. BLOTS
    • Southern
    • Northern
    • Western
  • 4. SOUTHERN
    • M.E Southern
    • ? Presence of restricfion fragments of DNA in a sample
    • Involves separation, transfer and hybrdization
    • Single gene/ larger piece of DNA.
  • 5. Process
    • 1) DNA is digested with a RE, separated by gel electrophoresis.
    • 2) Transferred to a membrane. Same pattern.
    • 3) The blot is incubated with many copies of a probe. This probe will form base pairs with its complementary DNA sequence and bind to form a ds DNA molecule. Radioactive/ enzyme bound (ALP or horseradish per).
    • 4) The location of the probe is revealed by incubating it with a substrate = colored product.
  • 6. Blotting
    • Nitrocellulose/Nylon paper laid over the gel
    • The gel is supported on a layer of sponge in a bath of alkali solution
    • Buffer is sucked through the gel and the nitrocellulose paper
    • As the buffer is sucked through, it denatures the DNA and transfers the single-stranded fragments from the gel to the surface of the nitrocellulose sheet. Adhere.
  • 7. Hybridization
    • The nitrocellulose sheet is peeled off the gel.
    • The sheet containing the bound ss DNA fragments is placed in a sealed plastic bag together with buffer containing a probe specific for the required DNA sequence. Exposure.
    • The sheet is removed from the bag and washed. Only probe molecules that have hybridized to the DNA on the paper remain attached.
    • Autoradiography = the DNA that has hybridized to the labeled probe will show up as bands on the autoradiograph.
  • 8.  
  • 9.  
  • 10. Closer look
  • 11. Gel electrophoresis
  • 12. Transfer of the DNA to a Membrane
    • The DNA is denatured –
    • Transferred onto a membrane.
    • Blotted
  • 13. Hybridization
    • Incubated with a soln
    • containing a probe. Comp.
    • A wash step removes any
    • probe which is not tightly
    • bound to the DNA on the
    • membrane.
    • Only DNA that matches
    • exactly will remain bound
  • 14. Northern blotting
    • RNA molecules of varying lengths are separated by size and blotted onto nitrocellulose.
    • A DNA probe (often a cDNA) is then used to identify bands that contain particular sequences.
    • Useful for determining the conditions under which specific genes are being expressed, including which tissues express which genes ?
  • 15. Western Blot
    • Proteins are separated by electrophoresis and blotted onto an appropriate support matrix.
    • The matrix is then exposed to an antibody to the desired protein and all unbound antibody is washed off.
    • The bands are detected by the binding of a second antibody that is radioactively labeled and specific for the first antibody.