Restriction Enzymes
Remember what we know about
DNA.
What is the monomer of DNA?
How do bases pair?
What kind of bond is used?
Restriction Enzymes
Aka Restriction
Endonucleases
What macromolecule do
you think they are made
of?
– Right, they are PROTEINS
that cut strands of DNA at
specific nucleotide
sequences
Restriction Enzymes
There are many different restriction
enzymes that each cut DNA at different
nucleotide sequences
Most will cut the DNA with a staggered cut
Usually occurs at a palindrome
5'GAATTC
3'CTTAAG
Sticky ends
The staggered cuts leave the DNA with
end pieces “sticking off”
– We call these “sticky ends”
– These exposed N-bases will want to join with
other complimentary exposed bases
What if???
What do you predict could happen if two
pieces of DNA are cut with the same
restriction enzyme???
– YES! They will have the same “sticky ends”
– How could we use this???
Restriction Enzymes -Kinds
Sticky End- already discussed
Blunt End
– These cut the DNA straight across and create
blunt ends:
– CCCGGG
GGGCCC
Products generated by restriction enzymes
COHESIVE END CUTTERS (staggered cuts):
Enzyme Recognition Site Ends of DNA After
Cut
EcoRI 5’…GAATTC…3’ 5’…G AATTC…3’
3’…CTTAAG…5’ 3’…CTTAA G…5’
PstI 5’…CTGCAG…3’ 5’…CTGCA G…3’
3’…GACGTC…5’ 3’…G ACGTC…5’
BLUNT END CUTTERS (direct cuts):
Enzyme Recognition Site Ends of DNA After Cut
HaeIII 5’…GGCC…3’ 5’…GG CC…3’
3’…CCGG…5’ 3’…CC GG…5’
In case you were curious …
Restriction enzymes are named according to the following
nomenclature:
Ex: EcoRI
E = genus Escherichia
co = species coli
R = strain RY13
I = first enzyme isolated
Why would anyone go through the
trouble of cutting DNA???
One reason…
– Recombinant DNA
Break down the word…what do you think
recombinant means?
– Other reasons…DNA fingerprinting, gene
therapy…
DNA that has been cut from one strand of
DNA and then inserted into the gap of
another piece of DNA that has been
broken.
– The host DNA is often a bacterial cell such as
E coli.
Bacteria are often used in biotechnology because
they have plasmids
A plasmid is a circular
piece of DNA that exists
apart from the
chromosome and
replicates independently of it.
The Plasmid is then called a
VECTOR
What is a vector?
– Something that is used to transfer genes into
a host cell
Ex’s
– Bacterial
plasmids
– Viruses
So how do I isolate a gene of
interest?
Use a restriction enzyme!!! (duh!)
What next???
Once the gene is isolated, how do we join
it with the organism’s DNA?
1. Cut the organism’s DNA with the same
restriction enzyme…why?
– The sticky ends will naturally be attracted to
each other
2. Add DNA LIGASE: an enzyme that seals
the fragments together
What is this organism now called?
Transgenic Organism- organisms that
contain functional recombinant DNA
(rDNA) from a different organism
What’s the point?
Recombinant DNA has been gaining importance over the
last few years, and will become more important as
genetic diseases become more prevalent and agricultural
area is reduced. Below are some of the areas where
Recombinant DNA will have an impact:
– Better Crops (drought & heat resistance)
– Recombinant Vaccines (i.e. Hepatitis B)
– Production of clotting factors
– Production of insulin
– Production of recombinant pharmaceuticals
– Plants that produce their own insecticides
– Germ line and somatic gene therapy
RECAP
Steps for making a
transgenic organism:
1. Locate and isolate the
gene of interest
2. Cut out the gene and cut
the plasmid using the
appropriate restriction
enzyme
3. Insert the desired gene into the plasmid
matching up the sticky ends
4. Use the enzyme DNA ligase to
seal up the sticky ends
5. Transfer the vector in the host organism
where it will replicate
6. Host organism produces the protein coded
for by the recombinant DNA
Insulin Production