1. Restriction Enzymes
Biotechnology
Fall 2013

2. Restriction Enzymes
Restriction enzymes are protein enzymes produced by bacterium
also known as 5’ – 3’restriction endonucleases
recognize a specific sequence of nucleotides in double-stranded DNA
Why?
What about self-mutilation?

3. History
In 1978 the Nobel prize was awarded to WERNER ARBER , DANIEL NATHANS and HAMILTON O. SMITH for the discovery of restriction enzymes and their application to problems of molecular genetics.
The first practical use of their work was the manipulation of E. coli bacteria to produce human insulin for diabetics

4. Restriction Enzymes
Each restriction enzyme is specific for a certain combination or sequence of nucleotides (their restriction site)
Scan DNA looking for the restriction site (the specific arrangement of nucleotides)
Once the recognition sequence is found, the DNA molecule is cut by the enzyme at that site in a predictable manner

5. Blunt vs. Staggered Ends
Restriction enzymes can leave blunt or staggered (“sticky”) ends at cutting sites 5’
CCC GGG
GGG CCC 3’ 5’
GAATTC
CTTAAG 3’
CCC GGG
GGG CCC
G AATTC
CTTAA G
Blunt ends
Staggered Ends

6.  Restriction Map Lambda bacteriophage
Very Well Know Restriction Sites

7.  DNA Marker Map
Measure the actual mobility (distance migrated) of the fragments seen in the picture of the gel. Measure from the bottom of the well to the mid point of the band

8.  Restriction Map
Make a graph of size vs.mobility of the DNA fragments on the semilog paper.
This is your standard curve.
Note: Do Not Connect Dots as in this picture, but make a Best Fit Line

9.  Restriction Map
Generate a  Hind III restriction map by labeling all bands in your picture of the gel
This will serve as a reference for other digests

10. Lab Experiment
We will be digesting Lambda DNA with three restriction enzymes:
HindIII
EcoR1
Pst1
The digest with HindIII will serve as the marker
We will find out how many EcoR1 and Pst1 restriction sites are in Lambda DNA, and what size fragments are created

11. Lab Experiment Steps Digest Lambda with the restriction enzymes
Make a standard curve of HindIII data
Use the standard curve of HindIII to analyze the digest with EcoR1 and Pst1