In the process of DNA cloning, a gene to be replicated is first obtained by treating the DNA molecule with restriction endonucleases and ligases (DNA, 1999). A gene can be obtained as a single restriction fragment after digestion of a larger molecule with a specific restriction enzyme. In this case, the DNA fragment comes from a virus. The next step is to prepare plasmids such as those from E.coli. The plasmids are cut in a single position, converting the normally circular DNA into a single linear strand with 5'-GAT-3' sticky ends. This plasmid DNA is now mixed with the viral DNA and ligases, and various recombinant ligation products are obtained, which consist of circular plasmid DNA with the viral DNA inserted into the position where the restriction site originally was.
The recombinant plasmid is now reintroduced into E. Coli and the plasmid plus the inserted gene will be replicated and copies will be passed on to daughter bacterial cells during cell division (DNA, 1999). This will give rise to a colony of E. Coli bacteria in which each bacterium contains multiple copies of the viral gene. To separate recombinant bacteria from non-recombinant bacteria, they can be plated on ampicillin plates containing X-gal. With most cloning vectors, including pUC8, the vector carries the ampicillin resistant gene from pBR322 and a second gene called lacZ, which is part of the E. Coli gene for the enzyme beta-galactosidase. The remainder of the lacZ gene is located on chromosome of the special E.Coli strain that is used when cloning genes with pUC8. Gene segments on the plasmid and the chromosome code for proteins which are able to combine to form functional beta-galactosidase enzyme molecule.
Beta-galactosidase molecules in the cells can be checked for functionality by a histochemical test with a compound, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal), which is converted to a blue product by the enzyme (DNA, 1999...