Explain the role(s) of the following in Biotechnology:

(a) Restriction endonuclease

(b) Gel – electrophoresis

(c) Selectable markers in pBR322.

(a): Restriction endonuclease- these are the enzymes that cut the DNA sequences at the specific site. These enzymes are used to form recombinant molecules of DNA, which are composed of PNR from different sources.

To insert and foreign DNA into an intact DNA, it must be cut from its sources and the intact DNA must also be cut open, both these process are carried by using the same restriction endonuclease.

(b): Gel-electrophoresis- it is a technique that allows us to separate DNA fragments on the basis of their size on an agarose gel matrix.

Since, the DNA are negatively charged molecule, they separate and move towards the anode (+ve) under the influence of an electric field.

(c): Selectable markers in pBR322- pBR322 is a widely used E.coli cloning vector. It has 2 antibiotic resistance genes, one for Ampicillin and the other one for Tetracycline. Antibiotic resistance is used as a selectable markers.

For example if the foreign DNA is ligated(joined) at the site of tetracycline resistance gene in pBR322 vecto, the recombinant plasmid will lose tetracycline resistance due to the insertion of foreign DNA, but can still be selected out from non-recombinants by plating the transformants on ampicillin-containing medium. The transformants grown on this medium is then transferred on a medium containing Tetracycline. The recombinants will grow in ampicillin containing medium but not on tetracycline-containing medium. However, the non-recombinants will grow in both the medium. Thus, by using antibiotic resistant genes as selectable markers, we can differentiate between recombinants and non-recombinants.