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Premium member Presentation Transcript Oligonucleotide Synthesis: Oligonucleotide Synthesis Bio-Resource www.technologyinscience.blogspot.com Oligonucleotides: Oligonucleotides Oligonucleotides are short, single-stranded DNA or RNA molecules that have a wide range of applications in genetic testing, research, and forensics. Oligonucleotide Synthesis: Oligonucleotide Synthesis Oligonucleotides are made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids can be manufactured with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR),DNA sequencing, library construction and as molecular probes. Solid Support for Oligo Synthesis: Solid Support for Oligo Synthesis The oligonucleotides are synthesized on solid supports from the 3’-end and the first monomer at this end is normally attached to a CPG(Controlled Pore Glass) or Polystyrene (PS). Solid Support for Oligo Synthesis: Solid Support for Oligo Synthesis Controlled Pore Glass (CPG) Controlled-pore glass is rigid and non-swelling with deep pores in which oligonucleotide synthesis takes place. Glass supports with 500 Å (50 nm) pores are mechanically robust and are used routinely in the synthesis of short oligonucleotides . However, synthesis yields fall off dramatically when oligonucleotides more than 40 bases in length are prepared on resins of 500 Å pore size. This is because the growing oligonucleotide blocks the pores and reduces diffusion of the reagents through the matrix. Although large-pore resins are more fragile, 1000 Å CPG resin has proved to be satisfactory for the synthesis of oligonucleotides up to 100 bases in length, and 2000 Å supports can be used for longer oligonucleotides . Solid Support for Oligo Synthesis: Solid Support for Oligo Synthesis Polystyrene (PS) Highly cross-linked polystyrene beads have the advantage of good moisture exclusion properties and they allow very efficient oligonucleotide synthesis, particularly on small scale (e.g. 40 nmol). Oligonucleotide Syntheis Method: Oligonucleotide Syntheis Method Phosphoramidite Synthetic Method McBride and Caruthers in 1983, developed this method of oligonucleotide synthesis. Process: Process De-protection, Coupling, Oxidation and Capping. Step1 - Deprotection: Step1 - Deprotection In the classic de-protection step the trityl group attached to the 5’ carbon of the pentose sugar of the recipient nucleotide is removed by trichloroacetic acid (TCA) leaving a reactive hydroxyl group. Step2 - Coupling: Step2 - Coupling In the coupling step, the phosphoramidite monomer is added in the presence of an activator such as a tetrazole , a weak acid that attacks the coupling phosphoramidite nucleoside forming a tetrazolyl phosphoramidite intermediate. This structure then reacts with the hydroxyl group of the recipient and the 5’ to 3’ linkage is formed . The tetrazole is reconstituted and the process continues. Step3 – Oxidation : Step3 – Oxidation The oxidation step stabilizes the phosphate linkage in the growing oligonucleotide. The traditional method of achieving this is by treatment with iodine in water. Step4 - Capping: Step4 - Capping The final step of the synthesis cycle is the capping reaction. Any remaining free 5’-hydroxyl groups are blocked at the capping step in an irreversible process. This step prevents the synthesis of oligonucleotides with missing bases. Following this step, the oligonucleotide is ready for the next monomer. Advantages of Solid Phase Synthesis : Advantages of Solid Phase Synthesis Large excesses of solution-phase reagents can be used to drive reactions quickly to completion Impurities and excess reagents are washed away and no purification is required after each step The process is amenable to automation on computer-controlled solid-phase synthesizers. Problems and Challenges: Problems and Challenges Monitoring coupling efficiency is critical parameter to get high yield of oligo synthesis. If the coupling efficiency is 99% then, theoretical yield for a 24mer will be 89.1% full-length product (FLP) at 99.5% average coupling efficiency and 79.4% FLP at 99.0% average coupling efficiency. Even a 0.5% average coupling failure rate can be dramatic for longer oligonucleotides . A minor increases in average couplingfailure rates will have a substantial net effect on even average length oligonucleotides . It is for this real-time monitoring of every custom synthesis reaction on every synthesis platform. Read More on: Read More on Bio-Resource www.technologyinscience.blogspot.com You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.