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 it’s made by combining DNA’s)

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