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![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](https://image.slidesharecdn.com/674bbc2c-8406-4e14-99eb-a07c68e87f21-150222120200-conversion-gate02/85/Restriction-Enzymes-12-320.jpg)
![Electrophoresis of SV40 DNA digested with H. influenzae restriction endonuclease. 2 ÎĽg 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](https://image.slidesharecdn.com/674bbc2c-8406-4e14-99eb-a07c68e87f21-150222120200-conversion-gate02/85/Restriction-Enzymes-13-320.jpg)
![Radioautographic analysis of SV40 DNA digested with H. influenzae restriction endonuclease.
1 ÎĽg of SV40 [14C]DNA I (3 Ă— 104 cpm/ÎĽg) was digested (see Fig. 2) for 6 hr in a volume of 55
ÎĽl; 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](https://image.slidesharecdn.com/674bbc2c-8406-4e14-99eb-a07c68e87f21-150222120200-conversion-gate02/85/Restriction-Enzymes-14-320.jpg)
Restriction Enzymes
- 1.
- 2. What are RestrictionEnzymes? â–ş Also referred to as Restriction Endonucleases â–ş Enters and recognizes a certain sequence on a double helix strand of DNA, usually 4-6 base-pairs long, and cuts it â–ş Precise by cutting both strands in same location though strands move in reverse directions; REs are able to depict the precise spot to cut â–ş Able to restrict and destroy foreign DNA, such as viruses, preventing them from entering the cell â–ş Used in biotechnology for cutting DNA into smaller strands for research in gene cloning or fragment lengths among different individuals
- 3. The Discovery ofRestriction Enzymes â–ş Restriction enzymes were first postulated by W. Arber in 1960 â–ş The first true restriction endonucleases was isolated in 1970 by Nathans and Smith â–ş All three scientists were awarded the Noble Prize for Physiology and Medicine in 1978 for the discovery of endonucleases Dr. Nathans
- 4. Dr. Daniel Nathanswith colleague, Dr. Hamilton Smith
- 5. Three Types ofRestriction Enzymes â–ş Type 1- Cuts DNA as far as 1000 base-pairs from the recognition site. Usually large enzymes with many subunits. â–ş 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. â–ş Type 3- Cuts approximately 25 base-pairs from the recognition site. Also very large with many subunits.
- 6. Type 2 RestrictionEnzymes ► 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.
- 10. Daniel Nathans andKathleen Danna Experiment â–ş Digested DNA from plague purified stocks SV40 with the restriction endonuclease Hemophillus influenza. â–ş Provided eleven fragments that were resolvable by polyacrylamide gel electrophoresis. â–ş Eight of which were equimolar to the original DNA. â–ş Fragments ranged from 6.5 Ă— 105 to 7.4 Ă— 104 daltons which was determined by electron microscopy, DNA content, or electrophoretic mobility.
- 12. Sucrose-gradient sedimentation ofSV40 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 SV40DNA 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 ofSV40 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 lengthsof 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 sizeand 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 analysisof 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(SimianVirus) 40? â–ş Small oncogenic virus meaning it is capable of forming tumors â–ş Contains double-stranded, covalently closed-circular DNA â–ş Monkey cells are the natural host of the virus â–ş Studied often due to its similarity to cancer cells and small genome â–ş Is 5243 base pairs in length
- 19. What is SV(SimianVirus) 40? â–ş In 1960, SV40 was isolated from normal monkey kidney cells, stocks of the Sabin poliovirus vaccine, and an adenovirus vaccine. â–ş 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 â–ş Research studies have not yet shown a specific direct link to cancer although it is suspected
- 20. Laboratory Applications for RestrictionEnzymes â–ş Provides different ways of manipulating DNA such as the creation of recombinant DNA, which has endless applications â–ş Allows for the large scale production human insulin for diabetics using E. coli, as well as for the Hepatitis B and HPV vaccines â–ş Cloning DNA Molecules â–ş 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, whichincludes 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.
- 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/