WHAT ARE RESTRICTION
ENZYMES?
Restriction Enyzmes
– molecular scissors
able to cut DNA
HOW DO RESTRICTION
ENZYMES WORK?
Usually cut DNA at a “palindrome” such as GAATTC.
Palindrome – word or phrase when spelled backwords,
spells the same word or phrase
Ex. BOB
MADAM I’M ADAM
A Toyota! Race fast, safe car. A Toyota
5’
3’
GAATTC
| | | | | |
3’ CTTAAG 5’
“Restriction site”
or
“Recognition Sequence”
HOW DO RESTRICTION
ENZYMES WORK?
RE’s cut DNA’s
phosphodiester bonds
and hydrogen bonds.
HOW DO RESTRICTION
ENZYMES WORK?
- RE’s generate two
different types of
“cuts”
- Sticky ends
- Blunt cuts.
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
amylo.
Strain
R
d
H
Order Isolated
I
III
I
Recognition Site
G^AATTC
A^AGCTT
BamHI
G^GATGC
HOW DO RESTRICTION
ENZYMES WORK?
Must provide correct temperature and
buffer (salt, pH) for enzyme to work.
Mimics cellular conditions of bacteria they
come from.
WHAT ARE RE’S USED FOR?
Genetic engineering –
pasting together DNA
from two different
organisms.
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.
RE Video
restriction enzymes.exe
Bill Nye on Restriction Enzymes
WHAT ELSE ARE RE’S USED
FOR?
Forensics – DNA Fingerprinting for crime
scene investigation and paternity testing.
Everyone’s DNA has a different sequence
– even though only 0.1% different.
How frequently
would
EcoRI
cut
DNA?
6
4 = once every 4096 bp
Lambda (48,514 bp) would expect about 12 EcoRI sites
THOUGHT QUESTION
Bacteria are prokaryotes.
Prokaryotes do not have a nucleus.
Both DNA and RE’s are in cytoplasm.
Why isn’t bacterial DNA cut by RE’s?
Methylation
See board
In humans, methyl groups are used to tag
genes to turn them on or off. Stay tuned.