Transcript Manipulating DNA

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