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 LigaseT4 DNA ligase – used to chemically join two pieces of

DNA together

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