1. Restriction Enzymes

2. Remember what we know about DNA.
What is the monomer of DNA?
How do bases pair?
What kind of bond is used?

3. 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

4. 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

5. 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

6. 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???

8. Restriction Enzymes -Kinds
Sticky End- already discussed
Blunt End
These cut the DNA straight across and create blunt ends:
CCCGGG
GGGCCC

9. 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’

10. Restriction enzymes are named according to the following nomenclature:
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

11. 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…

12. 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.

13. 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.

14. 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

15. So how do I isolate a gene of interest?
Use a restriction enzyme!!! (duh!)

16. 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

17. What is this organism now called?
Transgenic Organism- organisms that contain functional recombinant DNA (rDNA) from a different organism

18. 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

19. RECAP Steps for making a transgenic organism:
Locate and isolate the gene of interest
Cut out the gene and cut the plasmid using the appropriate restriction enzyme

20. 3. Insert the desired gene into the plasmid matching up the sticky ends

21. 4. Use the enzyme DNA ligase to seal up the sticky ends

22. 5. Transfer the vector in the host organism where it will replicate
6. Host organism produces the protein coded for by the recombinant DNA

23. Insulin Production