Gene are the important part of DNA that is responsible for particular trait in any organism. They are present naturally and is transferred as the hereditary unit of life.
Though genes are naturally present in any organism, but the development in science have made it possible to create gene under chemical environment. This may sound surprising, but the research work of scientist have made it possible to bring a breakthrough in the field of science.
Synthesis of gene artificially requires use of chemicals and certain technique that need to be employed. Hence, there are number of approaches that have been forwarded for chemical synthesis of gene in the specific laboratory environment. The three approaches forwarded for chemical synthesis of gene are:
1. Phosphodiester approach: In this approach, a linkage is formed between the phosphate groups of one nucleotide with the hydroxyl group of other nucleoside which is called ester linkage. This approach was the first idea for chemical synthesis of gene but unfortunately the formation of pyrophosphate and branching of oligonucleotides at internucleosidic phosphate was the chief downside in this approach.
2. Phosphotriester method: The major drawback that was faced in first approach also called phosphodiester approach was overcome in phosphotriester method by providing protection to phosphate group through ethylcyano group. Hence, the major difference between the two methods is that; in phosphodiester method; only two phosphoester linkage is present whereas in phosphotriester method, three phosphoester linkage is present that prevented the branching of oligonucleotides.
3. Phosphoramidite methods: This is a new and fresh approach for the preparation of oligonucleotides. Application of this approach gives long chain oligonucleotides reducing the effort and increasing the efficiency of the work. The use of phosphoramidite monomers and tetrazole catalysis makes it possible to synthesis long oligonucleotide chain. Hence, firstly it is important to synthesis Phosphoramidite, which is achieved by few steps of Deprotection, coupling, capping and stabilization.
- Deprotection: Deprotection here involves the removing of trityl group that is linked 5’carbon of receiver nucleotide, thus opening up volatile hydroxyl group for the addition of next base.
- Coupling: In steps of coupling; phosphoramidite nucleoside is attacked by the tetrazloe forming tetrazolyl phosphoramidite intermediate. This in turn reacts with hydroxyl group of nucleoside that is linked to CPG bead, thus resulting in formation of 5’- 3’ linkage. Therefore, this gives completely re-formed tetrazole.
- Capping: Acetic anhydride and N-methyl imidazole is added in the reaction mixture that caps those oligonucleotides that have failed to be coupled.
- Stabilization: It is the final step that involves stabilization of every added base by the process of oxidation.
Finally after having oligonucleotides, it is important to anneal them to form chemically synthesized gene, hence for this process ligase and polymerase is employed. In case of single- step PCR, the chemically synthesized oligonucleosides are first ligated and then the obtained gene is amplified using PCR. In two step PCR, ligase and polymerase are used to anneal those nucleotides that are obtained from first step PCR.
This topic is undoubtedly lengthy, that requires certain amount of time for proper understanding. Having a good knowledge on structure of DNA is very necessary to complete the assignment and projects on related topics of DNA.
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The main topics which are considered in Recombinant DNA Technology are discussed in brief as follows:
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