Restriction Enzymes Graham Churchwell

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

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

Dr. Daniel Nathans with colleague, Dr. Hamilton Smith

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.

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.

A Closer Look

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.

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

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

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

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

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

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

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

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

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

“First of all, many human

diseases are influenced by,

if not caused by mutations

in genes.” Daniel Nathans

“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

Thank you

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/

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/