Manipulating DNA

1
Manipulating DNA

• Biologists have tools to cut, separate, read and
  splice together DNA sequences

2
Cutting DNA

• Restriction enzymes
• Cuts DNA at a specific sequence
• Several hundreds of them are known
• Can be purchased from biochemical supply
  companies or can be proprietary

3
Separating DNA

• Agarose gel electrophoresis
• Separates by charge (DNA is negatively charged)
  and by fragment size

4
Reading DNA

• Sequencing gels
• Laser sequencing

5
Splicing DNA

• DNA cut by a restriction enzyme has a sticky
  end that will allow it to pair with another
  piece of DNA cut by the same restriction enzyme
• A DNA ligase enzyme patches the pieces of DNA
  together
• The joined pieces act like a single (but new)
  piece of DNA.
• The new DNA is called a recombinant DNA (because
  its made by combining DNAs)

6
Cell transformation

• New gene is inserted into a cell
• This changes the cells genetic makeup
• Prokaryotes and eukaryotes can be transformed

7
Transforming bacteria

• Bacteria have small circular DNA called Plasmids
  (in addition to their chromosome)
• Plasmids are separated from bacteria
• New DNA is spliced into the Plasmid
• Plasmid is put back into the culture for bacteria
  to take up
• Bacteria incorporate the Plasmid and make the
  protein that is coded for on the Plasmid (this is
  how Eli Lilly makes recombinant insulin)

8
Transforming eukaryotes

• Single celled eukaryotes like yeast are
  transformed the same way as bacteria because they
  have Plasmids
• Animal cells (insect and mammalian are harder and
  require a lot of work to insert the DNA into the
  chromosomes
•  Insect cells are transformed by insect viruses
  (baculovirus)
• Animal cells are transformed by lipofection
  (liposomes), electroporation, microinjection,
  fusion with a bacterial Plasmid, infection with a
  virus (vector)
• Epogen is made by transformed animal cells at
  Amgen