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

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

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

Reading DNA Sequencing gels Laser sequencing

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)

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

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)

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