TRANSFER AND CLONING OF THE INSULIN GENE
Legend:
Example of the technique of DNA cloning into a plasmid.: Insertion of the gene coding for insulin into a bacterial plasmid, which in turn carries the gene into a replicating bacterial cell that produces human insulin.
Plasmid: Plasmids are small circles of DNA found in bacteria cells, separate from the bacterial chromosome and smaller than it. They are able to pass readily from one cell to another, even when the cells are clearly from different species, far apart on the evolutionary scale. Consequently, plasmids can be used as vectors, permitting the reproduction of a foreign DNA by using the bacterial replicating system.
cDNA: Human genes composed of coding and non- coding sequences. The copy of the coding sequences is called cDNA. It can be obtained from the reverse transcription of messenger RNA.
The transcription and translation of the insulin cDNA will allow the production of a functional insulin molecule.
Transfer of the Insulin gene into a plasmid vector.
- The plasmid is cut across both strands by a restriction enzyme, leaving loose, sticky ends to which DNA can be attached.
- Special linking sequences are added to the human cDNA so that it will fit precisely into the loose ends of the opened plasmid DNA ring.
- The plasmid containing the human gene, also called a recombinant plasmid, is now ready to be inserted into another organism, such as a bacterial cell.
Cloning the Insulin gene
The recombinant plasmids and the bacterial cells are mixed up. Plasmids enters the bacteria in a process called transfection. With the recombinant DNA molecule successfully inserted into the bacterial host, another property of plasmids can be exploited - their capacity to replicate. Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several dozen copies.When the bacteria divide, the plasmids are divided between the two daughter cells and the plasmids continue to reproduce. With cells dividing rapidly (every 20 minutes), a bacterium containing human cDNA (encoding for insulin, for example) will shortly produce many millions of similar cells (clones) containing the same human gene.
From: BIO. "Biology in Perspective." Washington, D.C.: Biotechnology Industry Organization, 1990.