biotechnology biotechnology – manipulation of biological organisms (usually with dna itself) to...
TRANSCRIPT
Biotechnologybiotechnology – manipulation of biological organisms
(usually with DNA itself)
To study the functions of individual genes, molecular biologists will cut them out of a genome and place them into bacteria.
Isolating DNABefore DNA can be manipulated, it needs to be
isolated from the cells.
1. Cell membranes are disrupted use a detergent (lysis solution)
2. DNA precipitation ethanol used to dehydrate and aggregate DNA
3. DNA isolation / storage
Manipulating DNA
restriction endonucleases – enzymes that cut DNA backbones at specific sequences through hydrolysis
recognition site – the DNA sequence which restriction enzymes bind tounsure if enzymes scan DNA to find sequences
http://www.youtube.com/watch?v=8rXizmLjegI
Why Restriction Enzymes?
These enzymes are naturally found in bacteria.
Bacteria found to be resistant to some bacteriophage.
Restriction enzymes would cut viral DNA, not its own genome.
Recognition SitesRecognition sites are typically 4 to 8 bp in length.
They are always palindromic.
This sequence is specific for the EcoRI enzyme.
5’ G A A T T C 3’
3’ C T T A A G 5’
Restriction EnzymesRestriction enzymes are named according to the
organism from which it was identified.
Ex. EcoRI
E - Escherichiaco - coliR - strain RY13I - 1st enzyme in this strain
Restriction EnzymesBacillus amyloliquefaciens, strain H, 5th endonuclease
identified
Nocardia otitidis, 3rd endonuclease identified
BamHV
NotIII
Restriction Enzyme Cuttingsticky ends – enzyme digests (cuts) to make
overhangs
EcoRI 5’ G A A T T C 3’3’ C T T A A G 5’
5’ G 3’ 5’ A A T T C 3’
3’ C T T A A 5’ 3’ G 5’
5’ overhang
Restriction Enzyme Cutting
PstI 5’ C T G C A G 3’3’ G A C G T C 5’
5’ C T G C A 3’ 5’ G 3’3’ G 5’ 3’ A C G T C 5’
3’ overhang
Restriction Enzyme Cuttingblunt ends – enzyme digests to make straight ends
SmaI 5’ C C C G G G 3’
3’ G G G C C C 5’
5’ C C C 3’ 5’ G G G 3’
3’ G G G 5’ 3’ C C C 5’
Recombinant DNAComplementary sticky ends from different pieces of
DNA can be joined together – recombinant DNA
DNA MethylationInevitably, the organism which creates the
restriction enzyme will also have, in its genome, the sequence which will be cut.
methylases (methyltransferases) – a methyl functional group is added to the nitrogen base of a nucleotideREs do not recognize methylated basesmethyl groups can be used by the scientist to protect
DNA regions from being cut