How are oligonucleotides synthesized?

Oligonucleotides are typically made in the laboratory using phosphoramidite chemistry and solid-phase synthesis. This method begins by attaching a starting nucleoside to a solid support that is typically composed of polystyrene (PS) or controlled pore glass (CPG). Then the following protocol is used to synthesize oligonucleotides:

• Step 1. Detritylation - Before any additional nucleosides can be added, the 5'-4,4'-dimethoxytrityl (5'-DMT) protecting group must first be removed from the support-bound nucleoside.
• Step 2. Activation & Coupling – After detritylation the support-bound nucleoside is now primed to react with the next base in the oligo sequence, this is added as a nucleoside phosphoramidite monomer. The nucleoside phosphoramidite is dissolved in acetonitrile (ACN) and mixed with an activator (e.g. tetrazole) so that it can react with the 5'-hydoxyl group of the support-bound nucleoside.
• Step 3. Capping –  Any support-bound nucleosides with unreacted 5'-hydroxyl groups must be capped (i.e. blocked) or they will react during the next cycle resulting in an oligonucleotide with a missing base. Unreacted 5'-hydroxyl groups can be blocked using a mixture of two capping solutions consisting of acetic anhydride and N-methylimidazole (NMI).
• Step 4. Oxidation – The bond of the newly attached nucleoside phosphoramidite is then stabilized via iodine oxidation.
• Step 5. Detritylation – The 5'-DMT protecting groups is removed from the support-bound nucleoside chain. This allows the 5'-hydroxyl group to react with the next nucleoside phosphoramidite monomer. This cycle is repeated, once for every base to be added in the sequence, until the oligonucleotide is complete.
• Step 6. Cleavage – The finished oligonucleotide is removed or cleaved from the solid support.
• Step 7. Deprotection – Following cleavage, the oligonucleotide, which is dissolved in concentrated aqueous ammonia, is heated to remove protecting groups from the bases and phosphates.