Question

what procedure allows us to isolate and extract plasmid DNA and to avoid isolating the larger circular genomic-chromosomal DNA?

Why do many restriction enzymes require a 37 C temperature to be active and to hydrolyze DNA molecules while other restriction enzymes require 50 C temperature to be active and to hydrolyze DNA molecules?

Answer 1

Plasmid DNA is very frequently isolated by alkaline lysis method. In alkaline lysis method the plasmid containing cells pellet are re-suspended in EDTA containing buffer. The EDTA chelates the divalent cations and weekend the wall of cells. Further the cells are lysed by adding lysis buffer (SDS and NaOH). SDS lysed the cells and in this steps the proteins, large chromosomal DA as well as plasmid DNA gets denatured. Further neutralization with potassium acetate allows the separation of plasmid from large chromosomal DNA and proteins. Basically, neutralization with potassium acetate allows only plasmid DNA to reanneal and solubilised however the large chromosomal DNA gets precipitated. The plasmid may be collected/concentrated from the supernatant obtained after centrifugation.

The temperature for the optimal activity of restriction enzymes depends on bacteria from which the restriction enzymes have been isolated. Restriction enzymes that have been isolated from bacteria that can infect humans (EcoRI, from E. coli) demonstrate optimal activity at 37 C because these bacteria often live at 37C.   Therefore the enzymes from such bacteria have high activity at or around that temperature.

Enzymes coming from bacteria that live in other environments or extreme environments have maximum activity at high temperature. For example the TaqI, a restriction enzyme active at 65 C have been isolated from Thermus aquaticus, a thermophilic bacterium.