The synthesis of oligonucleotides is a chemical, no a biological-enzymatic process. In the enzymatic process of DNA synthesis, deoxy-ribonucleotide-5’-tri-phosphates are used as substrates during polymerization. In addition this process requires a primer and a template.
In the chemical synthesis of DNA, nucleotide substrates with a 3'-phosphate group, a large dimethoxytrityl (DMT) protective group attached to the 5’-hydroxyl (5’-OH), and additional protective groups attached to chemically-reactive base amines on the nucleotides, are used. Moreover, chemical synthesis does not require a primer or a template, meaning that the sequence of the single-stranded oligonucleotide can be arbitrarily chosen.
Step 1. Detritylation
Trichloroacetic acid removes the DMT group to free the 5'-OH for coupling.
Step 2. Activation
The 3' end of the next nucleotide monomer is activated with tetrazole. Tetrazole protonates the diisopropylamine moiety so that it becomes a good leaving group.
Step 3. Coupling
Nucleophilic attack by the free 5'-OH group on the activated monomer results in formation of the internucleotide linkage.
Step 4. Capping
Unreacted chains (failure sequences) are ‘capped' with an acetylating solution to prevent further elongation.
Step 5. Oxidation
The phosphite triester linkage is oxidized with iodine to the more stable phosphate triester form.
The most frequently used oligonucleotide synthesis strategy is based upon beta-cyanoethyl phosphoramidite chemistry.
The synthesis proceeds from the 3' to the 5' end of the oligonucleotide, which is in opposition to the enzymatically catalyzed reaction where polymerization proceeds from 5'- to 3'.
The 3'-nucleotide is bound to a solid glass or polystyrene support via the 3'-hydroxyl of the deoxyribose sugar. The 5'-OH is blocked with a DMT group.
Please note that there is no 3'phosphate group attached to this support-bound nucleotide. The synthesis cycle then proceeds as shown above.
The synthesis continues repeating this cycle until the sequence of the oligonucleotide has been built.