Biotechnology:Principles and Processes
Biotechnology deals with the techniques of using living organisms or enzymes from the organisms to yield products and processes that are useful to human. Genetic engineering is the manipulation of the genes in the organisms and the construction of a completely man-made gene, which functions as it is intended.Recombinant DNA technologyor RDT is an area of biotechnology which includes various methodologies by which foreign DNA is inserted into an organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry, etc. The new DNA formed by this process is called the recombinant DNA or rDNA.
The tools needed for the DNA recombinant technology are enzymes, cloning vectors and competent host cell. The techniques used in recombinant technology are gel electrophoresis, DNA elution, Southern blotting, gene transfer and cloning.
The first step in the recombinant technology is the identification and the isolation of the desired DNA. The isolated DNA is cut into several pieces by restriction enzyme and fragments are then separated on the basis of their size and charge by using gel electrophoresis. The desired DNA fragments are visualised on the gel by using a fluorescent dye. Southern blotting is a hybridisation technique that is used for the identification of a known DNA sequence in the given sample. Gene transferis the process of transfer of a desired gene from one organism into the cell of another organism. Two methods generally employed in gene transfer are indirect method anddirect method. The target gene is joined to the vector with the help of the DNA ligase to form a recombinant DNA molecule. The recombinant DNA molecules may be amplified billion times by the polymerase chain reaction.
The recombinant DNA is then introduced into a suitable host by various methods. The methods of transfer of the recombinant DNA into the host are transformation, transfection, electroporation, chemical method, microinjection and gene gun. After the insertion of the desired gene into the vectors, selection of the transformed cells is done by the help of genetic markers (for, e.g. Antibiotic-resistant genes) present in the vector. The gene product produced in the recombinant cell is extracted from it and purified.
Recombinant protein can be produced on a small scale, under laboratory conditions, inside the prokaryotic cells. Bioreactors are large culture vessels that are used to produce the desired protein on large scale. The cells containing the recombinant gene are grown in bioreactors and areready to be made into a consumable form and to be sold.