Name and explain the techniques used in the separation and isolation of DNA fragments to be used in recombinant DNA technology.
1. Fragments of DNA can be separated by a technique of gel electrophoresis.
2. Electrophoresis is a technique used for the separation of substances of different ionic properties. If such substances having different electric charges are taken in a solution and if an electric current is passed through this solution, the charged particles also move either towards anode or cathode, depending upon their charges, viz. positively charged through a medium. Since the DNA fragments are negatively charged molecules, they can be separated by allowing them to move towards anode.
3. Recently, the most commonly used matrix in gel electrophoresis is agarose which is polysaccharide extracted from sea weeds. DNA fragments move towards the anode according to their molecular size through the agarose gel. Thus the smaller fragments move farther away as compared to larger fragments.
4. The separated DNA fragments can be observed only after staining them with a solution of ethidium bromide. The bright orange coloured bands of DNA can be seen under UV light because pure DNA fragments cannot be seen under visible light.
5. The bands are cut out from the gel and extracted by using a convenient technique. This step is called elution. The eluted DNA fragments are then purified and used in constructing recombinant DNA by joining them with cloning vectors.