Biotechnology biotechnology – 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 DNA Before 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

DNA Isolation

Manipulating DNA restriction endonucleases – enzymes that cut DNA backbones at specific sequences through hydrolysis recognition site – the DNA sequence which restriction enzymes bind to unsure if enzymes scan DNA to find sequences

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 Sites Recognition 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 Enzymes Restriction enzymes are named according to the organism from which it was identified. Ex. EcoRI E- Escherichia co- coli R- strain RY13 I- 1 st enzyme in this strain

Restriction Enzymes Bacillus amyloliquefaciens, strain H, 5 th endonuclease identified Nocardia otitidis, 3 rd endonuclease identified BamHV NotIII

Restriction Enzyme Cutting sticky ends – enzyme digests (cuts) to make overhangs EcoRI5’ 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 PstI5’ 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 Cutting blunt ends – enzyme digests to make straight ends SmaI5’ 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 DNA Complementary sticky ends from different pieces of DNA can be joined together – recombinant DNA

DNA Ligase T4 DNA ligase – used to chemically join two pieces of DNA together

DNA Methylation Inevitably, 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 nucleotide REs do not recognize methylated bases methyl groups can be used by the scientist to protect DNA regions from being cut