If a desired gene identified in an organism for some experiments, explain the process of the following:
(i) Cutting this desired gene at specific location.
(ii) Synthesis of multiple copies of this desired gene.
(i) 1. Fragmentsof 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 substance having different electric charges are taken in a solution and if an electric current is passed through this solution, the charged particles also move eithers towards anode or cathode depending upon their charges, viz, positively charged molecules moves towards cathode of negatively charged molecules towards anode through the medium. Since the DNA fragments are negatively charged molecules, they can be separated by aloowing 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 through the agarose gel. Thus the smallest fragmants move farther away as compared to larger fragments.
4. The separated DNA fragments can be observed only after staning them with a solution of ethidium bromide. the bright orange coloured bands of DNA can be seen under UV light because pure DNA frogments cannot be seen under visible light.
5. The bands are cut out from the gel and extracted by using the convenint 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.
(ii) The technique to obtain multiple copies of a DNA segment synthesized in vitro is polymerase Chain Reaction.(PCR)
In this reaction multiple copies of the gene of interest are synthesized in vitro under three steps.
(i) Denaturation: In this double standerd DNA is coverted to single standerd, this is often achieved by heating or alkaline conditions. This is called melting of DNA.
(ii) Annealing: The two sets of primers (small chemical synthesized oligonucleotids that are complementary to the regions of DNA) undergo biochemical process of annealing at an optimum temperature of
(iii) Extension : The enzyme DNA polymerase extends the primers using the nucleotide provided in the reaction and the genomic DNA as template. If the process of replition of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times. Such repeated amplification is achived by the use of a thermostable DNA polymerces and the amplified fragment if desired can be used to ligate with a vector for further cloning.