Replied by Admin

ANSWER : In Recombinant DNA technology, recombinant DNA is made by combining DNA from two or more sources. DNA from a donor organism is first extracted from cells and then subjected to a cutting at specific sites by restriction enzyme. This generates fragments of DNA that contain the gene of interest. These fragments can then be cloned (or inserted) into recipient organism with help of vector such as plasmids, which are allowed to multiply gene of interest. The recombinant DNA is then recovered and verified. EXPLANATION : STEPS INVOLVED IN RECOMBINANT DNA TECHNOLOGY : 1) DNA ISOLATION : DNA must first be extracted and purified from cellular molecules, such as ribonucleic acids (RNAs), proteins, and structures such as cell membranes. For cloning purposes, DNA is obtained from the nucleus and is known as "genomic DNA." One common method for DNA extraction is by ultracentrifugation of cell components in a density gradient made up with ethidium bromide in cesium chloride. 2) RESTRICTION ENZYME DIGESTION OF DNA : Restriction enzymes are enzymes that cut up very specific DNA sequences; they are used to create unique DNA fragments. To generate the desired DNA fragments, a specific single (or combination of) enzyme(s) is used to cut up or digest the DNA. The fragments are then purified by gel electrophoresis, which separates them from the unwanted DNA. 3) DNA LIGATION : Ligation is the process of joining together the donor and recipient (or vector) DNA fragments to create a recombinant DNA molecule. As restriction enzymes produce "sticky ends" and ligase enzyme can be used to join the DNA segments with phosphodiester linkages. 4) RECOMBINANT DNA REPLICATION : The process of transformation or heat shock is used to put the recombinant DNA molecule into a host bacterial cell, which can then generate many copies of the synthetic DNA. These bacteria are grown on agar plates, cultured up in special bacterial broths, and then lysed in order to release the recombinant DNA. Finally, the DNA can be verified by DNA sequencing, functional experiments, and restriction enzyme digestion.

• 26 Oct 2017 @ 04:30