Microbiology Procedure

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Gene Cloning Procedure

	Gene Cloning Procedure Genetic engineering rests on two of the major discoveries of the last 20 years, namely, plasmids and restriction enzymes. Plasmids have already been described. We have already indicated about restriction enzymes also. They are in fact one of the bacterial defense systems against entry of foreign DNA. The restriction enzyme recognises a specific sequence of DNA bases and cuts the DNA at or close to that site. This specific sequences of bases occurs within the DNA of the bacterium but is protected from the action of the restriction enzyme by modification of one of the bases within the sequence. Foreign DNA is not similarly protected so that when it enters the cell it is rapidly degraded by the restriction enzyme.
The plasmids used in genetic engineering are relatives of the naturally occurring plasmids found in microorganisms. Generally they have been mutated and tailored with enzymes in order to have specific desired properties. Through genetic engineering (as an advantage over conventional genetic techniques) one can transfer a single specific gene between organisms. The donor DNA carrying the gene of interest is cut with a restriction enzyme to yield fragments of various sizes, one of which bears the desired gene. A suitable plasmid cut with the same enzyme, is mixed with the donor DNA and the two are joined by an enzyme, DNA ligase, to give a series of hybrid plasmids
The hybrid plasmid DNA is used to transform the host cell and the cells are plated out onto agar. The ratio of the amount of DNA to cells is adjusted in such a way that each cell takes up only one DNA molecule. Thus each colony which grows represents a different piece of the DNA donor carried on a plasmid. One of these carries the gene of interest and can be recognised by the characteristics it confers on the cell. This gene is then said to have been cloned This is the basis of genetic engineering. This method can be applied to DNA from any organisms and the host can be a bacterium, yeast, any other fungus or plant or animal cell. Genetic engineering has made significant contributions to industrial microbiology and in understanding the possible mechanisms of adaptability in microorganisms
In brief, gene cloning involves (i) isolation and fragmentation of the source DNA and incorporation of the fragments obtained into a cloning vector (segment of DNA used for replication of foreign DNA fragments), with the use of restriction endonucleases to cut and ligase to rejoin DNA molecules, (ii) incorporation of the genetically transformed DNA into a recipient organism that can replicate the cloning vector, (iii) detection of the newly transformed cells containing DNA and isolation of a pure culture there from and (iv) growth of culture of cells containing the cloned DNA fragment
	A cloning vector must be able to replicate autonomously in a suitable host. Plasmids are used as carriers of unrelated DNA for genetic engineering, and the enzymes used for splicing foreign DNA into the plasmid carriers are those used in normal recombination and replication of DNA. However, at the same time there are dangers also associated with this technology. There are possibilities of creating new and uncontrollable pathogens from tame microbes like E. coli and yeast.

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