Question

Describe various steps of recombinant DNA technique of genetic engineering.

Accepted Answer

The main technique of genetic engineering is recombinant DNA technology. The desired effective technique for manipulation of DNA of any organism is called recombinant DNA technology. Recombinant DNA is a modified DNA that is produced by the addition of a foreign gene into the basic DNA of an organism. This technique involves the following steps:

Identification and isolation of the desired gene: In this step, the desired gene is selected and is extracted from the cell with the help of various hydrolytic enzymes like protease, RNAase, lipase, etc. After extraction of DNA, the desired gene is isolated by cutting the DNA using restriction endonuclease enzymes. These enzymes cut the DNA at specific sites and make fragments. From the DNA fragments, the desired gene is selected by using a DNA probe.

Selection of cloning vectors: After isolation of the gene, a vector is required, which enters the host cell with the desired gene and replicates its DNA. Plasmid, bacteriophage, cosmid, transposons, etc., are used as a cloning vector.

Insertion of the desired gene in cloning vector: For the insertion of the desired gene into the vector DNA, the same restriction enzyme, which is used for cutting the desired gene, is used for cutting the vector DNA. Then the desired gene and vector DNA are ligated. The DNA ligase facilitates their ligation.

Multiplication of recombinant DNA in the host cell: The recombinant DNA is introduced into the host cells. E coli bacterium is generally used as the host cell.

Identification of the cloned gene and its transfer into other organisms: A special type of gene is used to select the recombinant DNA in the host cell. It is called a marker gene. It helps to identify and eliminate nontransformed cells and selectively allow the growth of the transformants. The genes encoding antibiotic resistance such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered as useful selectable markers for E. coli. The normal E. coli cells do not carry any antibiotic-resistant genes.

Expression of the desired gene: The ultimate aim of all recombinant technologies is to produce a desirable protein. The foreign gene gets expressed under appropriate conditions to get the desired product.

(a)	Bacillus thuringiensis	(i)	Cloning vector
(b)	Thermus aquaticus	(ii)	Construction of first rDNA molecule
(c)	Agrobacterium tumefaciens	(iii)	DNA polymerase
(d)	Salmonella typhimurium	(iv)	Cry proteins

Describe various steps of recombinant DNA technique of genetic engineering.

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