Genetic engineering is a highly specialized technique by which a DNA molecule (prokaryotic or eukaryotic) is broken at two desired places to isolate a specific DNA segment and then insert it in another DNA molecule at a desired position. The resultant DNA is called ‘recombinant DNA’. Using this technique, isolation and cloning of a single copy of a gene or a DNA segment into an indefinite number of identical copies has become possible. This process has become possible because bacteria, phages and phasmids reproduce in their usual manner even after the insertion of foreign DNA. The inserted DNA also replicates faithfully with the parent segment of choice has been developed called ‘Polymerase Chain Reaction’ (PCR). In PCR, a thermo-stable DNA polymerase (Taq polymerase) is used. The Taq polymerase is a DNA polymerase enzyme that is isolated from the bacterium Thermus aquaticus growing in the hot water springs. This enzyme acts best at 72°C and is not denatured at 90°C.
The process of isolation, cloning and transfer of desired gene or genes in the DNA of a desired organism is called “recombinant DNA technology”. Replication or cloning is possible only when the alien gene becomes part of the chromosome (genome) or extrachromosomal DNA (extragenomic DNA) which has its own origin of replication or “ori”. The “ori” is present in the plasmid. The plasmids are the extra-chromosomal, self-replicating circular DNA segment present in the bacterial cytoplasm. Cohen and Boyer (1972) first produced a recombinant DNA by attaching an ‘antibiotic resistance gene’ with the plasmid of the bacterium Salmonella typhimurium with the help of restriction endonuclease enzyme. The restriction endonuclease cuts the two strands of the DNA double helix at a specific region that has complementary sticky ends. The cut ends of the DNA double helix are joined together by ligase enzyme. The recombinant DNA is now forms multiple copies of the DNA. The organism with the recombinant DNA is called “transgenic” organism.