What are the important steps followed in Recombinant technology?

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Various steps in Recombinant technology are as follows:

At the first step, DNA molecules are cut into specific fragment containing identifiable genes by using restriction enzyme endonuclease.

Vectors are used for transferring the DNA fragment to a suitable recipient bacterium. Vectors may be plasmids, bacteriophages (viruses), cosmids and others. Auector facilitates the manipulation and recognition of newly created recombinant molecule. Plasmids are small, circular DNA molecules that occur naturally in bacteria. They carry genes for sexuality, antibiotic resistance etc. but not any vital gene so that a cell can survive even without them. Plasmid DNA replicates independently of the main genome and being small, can easily come out of or get into a cell.

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The restriction endonuclease used to cut the foreign DNS is also used to splice the plasmid at specific point in such a manner so as to have sticky ends in both the molecules.

The free sticky ends of the plasmid DNA and the foreign DNA serve as convenient points for their complementary pairing.

The gaps are the sealed by an enzyme ligase, thus making a circular DNA piece, which contains the plasmid genes, as well as a piece of foreign DNA. The product is referred to as recombinant DNA.

Such a recombinant DNA can be introduced into a bacterial cell as plasmid, where it can replicate and express itself. When such new foreign DNA fragments are introduced into host cells, such cells are said to be transformed and the process is known as transformation.

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Within the bacterial cell, the recombinant DNA molecule is replicated along with endogenous DNA of host cell during asexual reproduction a process referred to as cloning.

The cloned recombinant DNA produced in abundance in an overnight bacterial culture is isolated, purified and analyzed.

The foreign DNA can then be released from the recombinant plasmid of clone cells once again by the use of restriction enzymes. Thus, large quantities of foreign genes can be isolated by this technology. Potentially the cloned DNA can be transcribed its mRNA, translated and the gene product is isolated and studied.

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