. P ROCESSES OF R ECOMBINANT DNA T ECHNOLOGY 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. Let us examine each of these steps in some details. .
. Isolation of the Genetic Material (DNA) Recall that nucleic acid is the genetic material of all organisms without exception. 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. This can be seen as collection of fine threads in the suspension (Figure . ). Figure .
DNA that separates out can be removed by spooling