1. Restriction Enzymes
Graham Churchwell

2. What are Restriction Enzymes?
► Also referred to as Restriction Endonucleases
► Enters and recognizes a certain sequence on a double helix strand
of DNA, usually 4-6 base-pairs long, and cuts it
► Precise by cutting both strands in same location though strands
move in reverse directions; REs are able to depict the precise spot
to cut
► Able to restrict and destroy foreign DNA, such as viruses,
preventing them from entering the cell
► Used in biotechnology for cutting DNA into smaller strands for
research in gene cloning or fragment lengths among different
individuals

3. The Discovery of Restriction Enzymes
► Restriction enzymes were first postulated by
W. Arber in 1960
► The first true restriction endonucleases was
isolated in 1970 by Nathans and Smith
► All three scientists were awarded the Noble
Prize for Physiology and Medicine in 1978
for the discovery of endonucleases
Dr. Nathans

4. Dr. Daniel Nathans with colleague, Dr. Hamilton Smith

5. Three Types of Restriction Enzymes
► Type 1- Cuts DNA as far as 1000 base-pairs from the
recognition site. Usually large enzymes with many
subunits.
► Type 2- Most commonly used in biotechnology, they can
cut at desired location and do not require the need for
ATP. They are typically smaller than Type 1 and 3.
► Type 3- Cuts approximately 25 base-pairs from the
recognition site. Also very large with many subunits.

6. Type 2 Restriction Enzymes
► Type 2 enzymes have one of two kinds of cuts on
the strand of DNA.
► One is known as the “blunt end” cut, which has
no nucleotide overhangs.
► The other cut is a “sticky end” cut, which has an
overhang of nucleotides.
► Both cuts are useful in the making of recombinant
DNA and proteins.

8. A Closer Look

10. Daniel Nathans and Kathleen Danna
Experiment
► Digested DNA from plague purified stocks SV40 with the
restriction endonuclease Hemophillus influenza.
► Provided eleven fragments that were resolvable by
polyacrylamide gel electrophoresis.
► Eight of which were equimolar to the original DNA.
► Fragments ranged from 6.5 × 105 to 7.4 × 104 daltons
which was determined by electron microscopy, DNA
content, or electrophoretic mobility.

12. Sucrose-gradient sedimentation of SV40 DNA digested with H. influenzae restriction
endonuclease. 27 ng of SV40 [3H]DNA I (3.9 × 104 cpm/μg) was incubated at 37°C for 30 min
in 50 μl of TMSH–40 mM NaCl, either with no enzyme, with 1 μl (0.016 units) of enzym...
Danna K , and Nathans D PNAS 1971;68:2913-2917
©1971 by National Academy of Sciences

13. Electrophoresis of SV40 DNA digested with H. influenzae restriction endonuclease. 2 μg of
SV40 [32P]DNA I (8200 cpm/μg) in 0.09 ml of TMSH–50 mM NaCl was digested at 35°C.
Danna K , and Nathans D PNAS 1971;68:2913-2917
©1971 by National Academy of Sciences

14. Radioautographic analysis of SV40 DNA digested with H. influenzae restriction endonuclease.
1 μg of SV40 [14C]DNA I (3 × 104 cpm/μg) was digested (see Fig. 2) for 6 hr in a volume of 55
μl; 0.0015 unit of enzyme was added at 0 time and at 1, 2, 3, 4, and 5 ...
Danna K , and Nathans D PNAS 1971;68:2913-2917
©1971 by National Academy of Sciences

15. Histograms of lengths of digestion products A, B, C + D, and E, expressed as the percentage
of the length of SV40 DNA II on the same grid.
Danna K , and Nathans D PNAS 1971;68:2913-2917
©1971 by National Academy of Sciences

16. Relationship between size and electrophoretic mobility for SV40 DNA fragments produced by
cleavage with H. influenzae restriction endouncelease. —○—, percentage of total radioactivity
in each peak of the electropherogram of Fig. 2. —Δ—, percentage by length.
Danna K , and Nathans D PNAS 1971;68:2913-2917
©1971 by National Academy of Sciences

17. Sucrose-gradient sedimentation analysis of purified digestion products A and D. 80-μl
samples of purified products eluted from gels were sedimented for 4.5 hr in 5–20% neutral
sucrose gradients a described in the legend of Fig. 1.
Danna K , and Nathans D PNAS 1971;68:2913-2917
©1971 by National Academy of Sciences

18. What is SV(Simian Virus) 40?
► Small oncogenic virus meaning it is capable of
forming tumors
► Contains double-stranded, covalently closed-circular
DNA
► Monkey cells are the natural host of the virus
► Studied often due to its similarity to cancer cells and
small genome
► Is 5243 base pairs in length

19. What is SV(Simian Virus) 40?
► In 1960, SV40 was isolated from normal monkey
kidney cells, stocks of the Sabin poliovirus vaccine,
and an adenovirus vaccine.
► Subsequent analyses found that the Salk poliovirus
vaccine administered from 1955 to 1963 in the United
States was also contaminated with SV40, potentially
exposing an estimated 100 million people
► Research studies have not yet shown a specific direct
link to cancer although it is suspected

20. Laboratory Applications for
Restriction Enzymes
► Provides different ways of manipulating DNA such
as the creation of recombinant DNA, which has
endless applications
► Allows for the large scale production human insulin
for diabetics using E. coli, as well as for the
Hepatitis B and HPV vaccines
► Cloning DNA Molecules
► Studying nucleotide sequence

21. “First of all, many human
diseases are influenced by,
if not caused by mutations
in genes.”
Daniel Nathans

22. “Small science, which includes most
research in the life sciences all over the
world, is science directed usually by an
individual senior scientist and a small team
of junior associates of three, ten, fifteen,
something in that order.”
Daniel Nathans

23. Thank you

24. Citations
1. http://what-when-how.com/molecular-biology/sv40-
simian-virus-40-molecular-biology/
2. http://medical-
dictionary.thefreedictionary.com/oncogenic+virus
3. http://biotech.about.com/od/proteinengineering/a/restricte
nz.htm
4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC153983/

25. Citations
5. http://www.youtube.com/watch?v=aA5fyWJh5S0&l
ist=PLMPpzatofcoFv2nXouJAYeoEfuz3Y_Vg7&in
dex=2
6. http://www.youtube.com/watch?v=zIS-E5WCmOE
7. http://www.authorstream.com/Presentation/Kiran25-
1404370-restriction-enzyme-endonucleases/