Southern Blot


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Southern Blot

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