Genetic engineering may be defined as construction and utilisation of new DNA molecules that have been engineered by recombinant DNA techniques. The technique of genetic engineering is in the production of recombinant DNA.
Recombinant DNA, as the name suggests, involves cutting a piece of original DNA and inserting in its place a different segment of DNA having desired characters. The recombined or recomposed DNA is then copied multifold inside bacterial cells and stored in a gene library for use when required. The multiple copies of the gene are
termed cloned DNA or cloned genes.
Recombinant DNA technology resulted from the two discoveries made while experimenting with bacteria :
(i) presence of plasmids or extra chromosomal DNA fragments in the bacterial cell which replicate along with bacterial DNA and can be used as a vector for carrying foreign DNA.
(ii) presence of specific restriction enzymes which attack and cut DNA at specific sites.
PROCESS OF RECOMBINANT DNA TECHNOLOGY
Recombinant DNA technology is a “cut and paste” technology. Specific nucleotide sequences are cut from the DNA of humans, other animals or plants and “pasted” into plasmids. DNA of the plasmid carrying nucleotide sequence of another organism
is the recombinant DNA. It is then inserted into bacteria. Bacteria divide repeatedly and a clone of bacteria with the recombinant DNA is obtained.
REQUIREMENTS FOR RECOMBINANT DNA TECHNOLOGY
(i) Cell culture (ii) Restriction endonuclease enzyme
(iii) Plasmids (iv) Ligases
(v) Host bacteria
(i) Cell culture : Cultured cells of an animal or plant (or even a bacterium) carrying the required gene (nucleotide sequence of DNA) in its nucleus.
(ii) The enzyme Restriction endonuclease : Restriction endonucleases cut short specific DNA sequences. There are many different restriction endonucleases found in bacteria. Each of these enzymes very specifically recognises a particular DNA sequence (usually 4 to 6 bases) and cuts it. These enzymes are the “molecular scissors”. They either cut both the strands at the same place or at different places so that the two DNA strands hang out at the two ends. Two cuts at the two ends of a DNA segment releases the cut part as the restriction fragment. The ends are single stranded and called sticky ends. Thus a piece of DNA containing a particular gene can be obtained by selecting a particular restriction endonuclease.
(iii) Plasmids : Plasmids are extra chromosomal DNA molecules in a bacterial cell which have sequences matching those of the required gene and can be similarly cut by the same restriction enzymes. Plasmids can readily enter bacteria, yeast or other speedily reproducing cells.
(iv) DNA ligase : It is an enzyme called ‘joining enzyme’ since it joins two DNA fragments, both of which have having sticky ends. Ligase is the “molecular glue”.
(v) Host Bacteria : Host bacteria are the bacteria whose plasmid is used for carrying foreign DNA.
STEPS IN RECOMBINANT DNA TECHNOLOGY
1. Specific restriction enzyme is selected.
2. Cell culture with required gene in the cells is obtained.
3. Restriction enzyme cuts the DNA at two ends of the specific gene and a restriction fragment is obtained
4. Same restriction enzyme cuts a matching DNA sequence from a plasmid
5. Ligase joins the restriction fragment in the place vacated by the cut DNA segment of the plasmid. The plasmid becomes a recombinant plasmid containing a foreign DNA fragment . Its DNA is the recombinant DNA. Since plasmids can carry foreign DNA, they are called clonal vectors. Bacteriophages (viruses) can also function as clonal vectors.
6. The recombinant plasmids are then placed with the comptent cells to enter the bacteria.
7. Bacteria divide. Recombinant plasmids replicate along with bacterial DNA.
8. A large population of bacteria (more than a million) containing recombinant DNA can be obtained in less than ten hours.
9. Multiple identical copies of DNA fragments inserted into plasmids or bacteriophage (bacterial virus) are then obtained and preserved in a DNA library.
10. These DNA fragments are the cloned DNA.
USES OF RECOMBINANT DNA TECHNOLOGY
1 . Recombinant DNA is widely used in biotechnology, medicine and research.
2 . The most common application of recombinant DNA is in basic research, in which the technology is important to most current work in the biological and biomedical sciences.
3 . Recombinant DNA is used to identify, map and sequence genes, and to determine their function.
4 .Recombinant DNA probes are employed in analyzing gene expression within individual cells, and throughout the tissues of whole organisms.
5 . Recombinant proteins are widely used as reagents in laboratory experiments and to generate antibody probes for examining protein synthesis within cells and organisms.
6 . Many additional practical applications of recombinant DNA are found in industry, food production, human and veterinary medicine, agriculture, and bioengineering. Some specific examples are – Insect-resistant crops , Recombinant hepatitis B vaccine , Golden rice , Recombinant human insulin , Recombinant human growth hormone , Recombinant blood clotting factor VIII , etc .