(a) Restriction endonuclease: These are enzymes that make cuts at specific positions within the DNA. They bind to their specific recognition sequence and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones. They are used in biotechnology to form recombinant molecules, which are composed of PNR from different sources. To insert a foreign DNA into an intact DNA, it must be cut from its source and the intact DNA also must be cut open. Both these Processes are carried by using the same restriction endonucleases.
(b) Gel electrophoresis: It is a technique that allows us to separate DNA fragments on the basis of their size. In this technique, the negatively charged DNA fragments are forced to move under an electric field through a medium or matrix. all the DNA fragments thus move towards anode and get separated according to their size through sieving effect provided by the agarose gel. The DNA of desired length can then be isolated and used in constructing recombinant DNA by joining them with cloning vectors.
(c) pBR322 has two antibiotic resistance genes, one for ampicillin and other one for tetracycline. Antibiotic resistance serves as selectable marker. If the foreign DNA is ligated at the site of tetracycline resistance gene in pBR322 vecto, the recombinant plasmid will lose tetracycline resistance due to insertion of foreign DNA but can still be selected out from non-recombinants by plating the transformants on ampicillin-containing medium. The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline. The recombinants will grow in ampicillin containing medium but not on the tetracycline-containing medium. However, the non-recombinants will grow on both. Thus by using antibiotic resistant genes as selectable markers, we can differentiate between recombinants and non- recombinants
