Recombinant DNA

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Recombinant DNA

• rDNA

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rDNA

• contains DNA from 2 or more different sources
• the DNA has been spliced together!
• See page 501 (Mader)

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Making rDNA

• animation of "sticky ends"

?Need 2 Enzymes 1. Restriction Enzymes
(molecular scissors) -a specific restriction
enzyme will cut DNA at a specific cleavage site
(a sequence of bases)
animation of section of DNA inserted into a
plasmid
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This is known as a Restriction Site

• Restriction enzymes recognize a specific short
  nucleotide sequence

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2. DNA ligase

• - seals any breaks in the DNA molecule.
• ?Need a VECTOR
• (something to carry the DNA into a host cell)
• -we often use bacterial
• PLASMIDS (small,
• circular pieces of bact. DNA)

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The Procedure

• 1. A bacterial plasmid is isolated.

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• 2. The desired human gene is cut out of the
  human DNA using restriction enzymes (example of a
  restriction enzyme is EcoR1.)

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• 3. A bacterial plasmid is also cut with the
  SAME restriction enzyme. (this leaves the human
  DNA and the plasmid DNA with the same sticky
  ends)

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• 4. The two kinds of DNA are mixed together with
  some DNA ligase.

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• 5. Bacteria are mixed with the newly engineered
  plasmids. (they readily take up plasmids -
  especially if you treat them with CaCl2, which
  makes them more porous)

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• 6. Bacteria are allowed to replicate.

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• 7. Bacteria are tested for the presence of the
  human gene.

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• NB The gene that is inserted into the plasmid
  will only work if it DOESNT have any introns.
  One way to do this is to synthesize the gene in a
  machine. Another method is to isolate the mRNA
  for the gene and use REVERSE TRANSCRIPTASE to
  make a DNA copy of it.
• ( complementary DNA (cDNA))

v. detailed animation of the whole process
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Uses for rDNA

• Mass production of hormones ,proteins, and
  chemicals
• ex. Insulin

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• 2. Producing safer vaccines.
• Eg. Hepatitis B vaccine

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• 3. Producing Transgenic plants and animals
• pest -resistant crop species
• larger cows (they produce more growth -hormone)

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Farms in the Future?
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• 4. Gene Therapy (See page 508, Mader)
• - Ex vivo (outside the body)
• Cells from the patient are given normal genes to
  replace defective ones, then the cells are
  returned to the patient to replicate and grow.

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• In vivo (inside the body)
• eg. A virus carrying a normal gene is inhaled by
  the patient. The virus is able to provide the
  patient with the normal gene product that the
  patient was missing due to a genetic disorder.