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Process of Recombinant DNA Technology - I

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Process of Recombinant DNA Technology - I - Lesson Summary

Recombinant DNA technology is a sophisticated process involving many steps. In order of sequence, these steps are isolation of genetic material or DNA, fragmentation of DNA by restriction endonucleases, amplification of the gene of interest, insertion of recombinant DNA into a host cell or organism, obtaining the foreign gene product and downstream processing. The genetic material in most organisms comprises DNA. In recombinant DNA technology, the first step is to select and isolate the piece of DNA of interest to be inserted into a cloning vector. Then, this piece of DNA is cut using restriction enzymes. To cut with restriction enzymes, the DNA should be in a pure form. That is, it should be free of macro molecules. To obtain DNA, a sample of cells is generally blended or ground thoroughly. This helps separate the cells from each other. Now, in order to isolate DNA enzymes such as lysozyme, cellulase and chitinase are used to treat bacterial cells, plant cells and fungal cell respectively. DNA is enclosed in the cell membranes along with macromolecules such as RNA, lipids, polysaccharides and proteins. Genes are located on long chains of DNA, which are intertwined with proteins such as histones. These proteins can be removed by treating them with protease, an enzyme used to break down proteins. On the other hand, RNA can be removed by treating it with ribonuclease. Finally, after the addition of chilled ethanol, DNA is isolated as a precipitate. This extracted DNA can be collected using a method called spooling. After isolation of DNA, in the next stage, restriction enzymes are used to cut the purified DNA molecules at specific locations. This cutting results in DNA fragmentation. The fragmented DNA can be separated according to its molecular weight by using a technique known as ‘gel electrophoresis’. It is employed to check the progression of restriction enzyme digestion. The bigger the DNA fragment, the slower it moves towards the positive pole. Therefore, smaller fragments reach the bottom of the gel first.  Stained agarose gel with DNA fragments can be seen under the UV light chamber. Later, the cut out piece of the source DNA or the foreign DNA fragment and the cut plasmid DNA are joined with the help of enzyme DNA ligase. These first three steps —isolation of DNA, fragmentation by restriction endonuclease and amplification of gene of interest—are important steps in recombinant DNA technology that are carried out using specific techniques.


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