Automatic solid phase oligonucleotide synthesis features simple operation, efficiency, and reproducibility. Almost all commercially available automated synthesis instruments are designed to support the phosphoramidite method for the chemical synthesis of nucleic acids. During this chemical synthesis, a phosphoramidite is coupled through its reactive 3′-phosphorous group to the 5′-hydroxyl group of a nucleoside immobilized on a solid phase. Completion of a single synthetic cycle results in the addition of one nucleotide residue to the growing chain. Each addition cycle consists of four chemical reactions including detritylation (or called deblocking), coupling, capping, and oxidation.
In the detritylation step, the dimethoxytrityl (DMT or “trityl”) protecting group at the 5′-terminus is removed by adding an acid solution to release a free 5′-hydroxyl group on the oligonucleotide precursor. Following the detritylation, the resulting nucleoside reacts with the incoming nucleoside phosphoramidite monomer via a coupling activator. Despite many efficiency measures, a small percentage of the unreacted 5′-hydroxyl sites remain active after the completion of the coupling step. In the capping step, any unreacted active 5′ hydroxyl groups of nucleotides are capped by undergoing acetylation using acetic anhydride and N-methylimidazole (NMI) to prevent the groups from reacting with the later phosphoramidites. The phosphite triester linkage formed in the coupling step is unstable and must be transferred to a more stable phosphate triester linkage by treating the growing oligonucleotide chain with oxidation agents. By repeating the synthetic cycle, the chain grows to the desired oligonucleotide, which can be released from the solid support and deprotected with the ammonium hydroxide.
Amerigo Scientific offers high-quality ancillary reagents covering each step of the oligonucleotide synthesis cycle. From deblocking to oxidation, we ensure that our ancillary reagents are suitable for each synthesizer type. These products include drying traps, acetonitrile diluent and washings, deblock solutions, activators, oxidizers, sulfurizing reagents, capping reagents, and ancillary reagents for H-phosphonate chemistry.