Biology BIOTECHNOLOGY - PRINCIPLES AND PROCESSES

Gene transfer method

Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. In order to force bacteria to take up the plasmid, the
bacterial cells must first be made ‘competent’ to take up DNA. This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 420C (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.
This is not the only way to introduce alien DNA into host cells. In a method known as micro-injection, recombinant DNA is directly injected
into the nucleus of an animal cell. In another method, suitable for plants, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in a method known as biolistics or gene gun. And the last method uses ‘disarmed pathogen’ vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host.
Now that we have learnt about the tools for constructing recombinant DNA, let us discuss the processes facilitating recombinant DNA technology.

PROCESSES OF RECOMBINANT DNA TECHNOLOGY

Recombinant DNA technology involves several steps in specific sequence such as isolation of DNA, fragmentation of DNA by
restriction endonucleases, isolation of a desired DNA fragment, ligation of the DNA fragment into a vector, transferring the
recombinant DNA into the host, culturing the host cells in a medium at large scale and extraction of the desired product.

Isolation of the Genetic Material (DNA)

In majority of organisms this is deoxyribonucleic acid or DNA. In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macro-molecules. Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).

You know that genes are located on long molecules of DNA interwined with proteins such as histones. The RNA can be removed by
treatment with ribonuclease whereas proteins can be removed by treatment with protease. Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol.