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Attachment of the first nucleotide to a solid support. The most common ones are polystyrene and controlled pore glass (CPG or PS). This is the starting point of the growing chain.

After the removal of the 5’-DMT protecting group from the support-bound 3’-nucleoside, detritylation, the next base is ready to be added. An excess of appropriate nucleoside phosphoramidite monomer gets protonated through dissolving with an activator (tetrazole) in acetonitrile. The process allows the coupling of 5’-hydroxyl group of the support-bound nucleoside to the new nucleotide forming a phosphite-triester.

To prevent unreacted 5’-hydroxyl group of the support-bound nucleoside to partake in the next coupling step, capping is required to block the 5’-hydroxyl group. Acetic anhydride and N-methylimidazole (NMI) combine to form activated acetic acid, which is used for acetylation of the 5’-hydroxyl group. ​

The bond of the phosphite-triester from the coupling step is unstable to acid and needs to be strengthened through iodine oxidation before the next detritylation step. Detritylation happens again to remove the 5’-end of the support-bound DNA chain so the primary hydroxyl group can bind with the next nucleotide. The cycle will repeat for each base until the required sequence is met. ​

The linker that attaches the 3’-end of the oligo to the support group and the most frequently used in synthesis is succinyl linker. This linker can be cleaved by incubating in ammonium hydroxide for one hour at room temperature. This step can be done on some synthesizers or manually by removing the column from the synthesizer and washing it with ammonium hydroxide.​

To get ready for purification, the oligonucleotide is heated in the concentrated aqueous ammonia from previous step to remove protecting groups from the heterocyclic bases (A, C & G), and the phosphodiester backbone (protected as 2-cyanoethyl phosphotriesters). ​