Download RESTRICTION ENZYMES

WHAT ARE RESTRICTION
ENZYMES?
Restriction Enzymes
– molecular scissors
able to cut DNA
nuclease
Enzyme digests nucleic acids
WHAT DNA SEQUENCES DO
RESTRICTION ENZYMES CUT?
Usually cut DNA at a “palindrome” such as
GAATTC.
Palindrome – word or phrase when spelled backwords,
spells the same word or phrase
5’
3’
GAATTC
Ex. BOB
| | | | | |
“Restriction site”
MADAM I’M ADAM
or
3’ CTTAAG
5’
A Toyota!
Race fast, safe
car. A Toyota
“Recognition Sequence”
WHAT PARTS OF DNA ARE CUT?
RE’s cut DNA’s
phosphodiester bonds
and hydrogen bonds.
WHAT’S LEFT AFTER RESTRICTION
ENZYMES CUT DNA?
- RE’s generate two
different types of “cuts”
- Blunt cuts.
- Sticky ends
WHERE DO RE’S COME FROM?
Bacteria
“Immune system” to
protect against
bacteriophages
(bacteria-infecting
viruses like Lambda).
HOW ARE RE’S NAMED?
After bacteria which produces them.
EcoRI
HindIII
Genus
Escherichia
Haemophilus Bacillus
Species
coli
influenzae
Strain
R
d
H
Order Isolated
I
III
I
Recognition Site
G^AATTC
A^AGCTT
BamHI
amyloliquefaciens
G^GATCC
WHAT MUST BE AVAILABLE FOR
RESTRICTION ENZYMES TO WORK?
All enzymes need the correct:
1. temperature
2. pH
“buffer”
3. salt concentration
Why are enzymes so needy? Need the
same environment as the cell that
produced them.
WHAT ARE RE’S USED FOR?
Genetic engineering –
pasting together DNA from
two different organisms.
Examples:
Bacteria with human insulin gene
Golden Rice with Vitamin A gene
Cows with human milk protein genes
Goats with spider silk genes
WHAT ARE RE’S USED FOR?
Forensics (not always
crime scenes)
Examples of Transgenic
Organisms
“Transgenic” – moving genes across species
Examples:
Bacteria with human insulin gene
Golden Rice with Vitamin A gene
Cows with human milk protein genes
Goats with spider silk genes
HOW DO RESTRICTION
ENZYMES WORK?
Which are more useful in genetic
engineering? RE’s that generate sticky ends
or ones that produce blunt cuts?
STICKY ENDS
HOW IS DNA PASTED
TOGETHER?
Ligase – another enzyme which
reconnects phosphodiester bonds.