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Recombinant DNA Techniques by cuiliqing

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									     Recombinant DNA
        Techniques
       Playing god for fun and profit!
                     Or
What happens when a mummy test tube and
a daddy test tube love each other very much.
  What is Recombinant DNA?
• DNA from one organism that has had DNA
  from another organism inserted in it. It
  may be from a different species.
                   Tools
• Really it is just cut and paste job using
  chemicals instead of scissors and glue.
• The scissors are Restriction enzymes.
• The glue is called DNA Ligase.
The Scissors: Restriction Enzymes
• Restriction Enzymes are enzymes that cut
  DNA at a specific sequences of bases.
• Some will make a straight cut so the cut
  ends of the DNA are even in length.
• Others will make a jagged cut with one
  strand longer than the other; this is known
  as a “sticky end”.
EcoRI digestion produces "sticky" ends,




   whereas SmaI restriction enzyme
    cleavage produces "blunt" ends
      The Glue: DNA Ligase
• DNA ligase is used to glue the cut ends of
  DNA together.
• Works with both sticky and blunt ends but
  sticky ends produce a better yield.
• Why would you use blunt ends?
 Let’s see the cutting and pasting
          process in full:
• http://www.youtube.com/watch?v=aA5fyW
  Jh5S0
                      Vectors
• Once you have created your recombinant DNA
  you then have to find a way to stick it inside your
  organism.
• How you do this will depend on:
   – What organism you want to put it in.
   – Whether you want the genetic change to be
     permanent
   – If you want the change to be pass on the offspring
   – How precisely you want to place the DNA in the
     genome
                 Bacteria
• Plasmids
  – Bacteria naturally have small circles of DNA
    with genes on them (these is different from
    their main chromosomal DNA) that they may
    pass between themselves.
  – Recombinant plasmids, as seen in a previous
    slide can be put into bacteria to give them
    new genes.
  – They may be inserted into bacteria using
    various methods.
              Eukaryotes
• Viral vectors
• Yeast artificial chromosomes.
• Gene guns
 Identifying Cells with Recombinant
                 DNA
• In bacteria:
   – add antibiotics resistance genes to the recombinant
     plasmid.
   – add a marker gene that causes a colour change.
• Use antibodies to detect protein of inserted gene.
• Complementary RNA or DNA probes (these are
  probes that have DNA that is complementary to
  your inserted genes that have a marker (e.g.
  florescent dye) on them).

								
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