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Date Posted: 11:46:16 08/01/04 Sun
Thermostability of Various Restriction Enzymes
By Alex Taylor
How well will three restriction enzymes with known and different optimal temperatures cleave ë DNA at different temperatures?
SmaI cleaved the ë DNA at room temperature but not 37°C or 70°C. EcoRI and HindIII cleaved at room temperature and 37°C but not at 70°C.
1. Incubate ë DNA, 2X restriction buffer, SmaI, EcoRI, and HindIII separately at room temperature for 10 minutes.
2. Add the reagents quickly but accurately in the following way:
Tube Ë DNA Buffer SmaI EcoRI BstBI H20
S 4ìl 5ìl 1ìl - - -
E 4ìl 5ìl - 1ìl - -
H 4ìl 5ìl - - 1ìl -
C 4ìl 5ìl - - - 1ìl
3. Pulse tubes in microfuge
4. Incubate tubes for 30 minutes at room temperature
5. Cast 0.8% Agarose Gel and set up electrophoresis box
6. Add 1ìl of loading dye to each tube (S, E, B, C) and pulse in a microfuge
7. Load contents of tubes into separate wells and electrophorese for 40-60 minutes.
8. Repeat steps 1-7 with incubation at 37°C.
9. Repeat steps 1-7 with incubation at 70°C.
IV. This experiment shows that different restriction enzymes have not only different optimal temperatures but different degrees of thermostability. By looking at their thermostability range, one can deduce where the enzyme occurs in nature. The SmaI only functioned at low temperatures (room temperature). This probably means that SmaI does not work inside the bodies of animals, which are warm. HindIII and EcoRI work well at 37°C, which is about body temperature. This means that they are probably found inside animals, and specifically mammals, which keep a regulated temperature around 37°C. None of the enzymes work well at 70°C, which was to be expected, because that is a very high temperature. HindIII works the best at 70°C, as there was some cleavage of the ë DNA. This probably means that HindIII exists in areas with higher temperatures, so it probably exists in a bacteria that causes fever. This experiment has shown that different restriction enzymes will work better at different temperatures, and thus are suited best for different jobs. Bacteria that produce restriction enzymes that are well suited for the temperatures that they live in will meet with more success, so as a result restriction enzymes are found in bacteria that live at the temperatures that those restriction function best at. This occurs through natural selection, and allows us to deduce the nature of their origin, or at least the temperature of their origin by their thermostability. When I researched this, I found that in fact HindIII is found in Haemophilus influenzae, a bacteria that causes illness. EcoRI is found in E. coli, which is found in human intestinal tracts. SmaI is found in Serratia marscescens, a bacteria that frequently grows on bread in damp places. It also grows in animals, but its main niche is room temperature food. By looking at the thermostability of various restriction enzymes, it was not hard to deduce vaguely what kind of bacteria it belonged to, and research vindicated all of the guesses. If looking for the origin of a restriction enzyme, thermostability is not a bad place to start.
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