Fig. 3. Schematic showing step-wise oligonucleotide synthesis via phosphoramidite solid-state chemistry. Synthesis is carried out by the addition of one nucleotide per coupling cycle, as shown here. Step 0: the dimethoxytrityl (DMT) protective group of the 3′end base that is bound to the controlled pore glass bead (CPG) is removed using deblocking reagents such as 3% trichloroacetic acid in dichloromethane. Step 1: The same process occurs during synthesis in which the DMT group on the last nucleotide attached to the CPG-oligo is deprotected. Step 2: Representative structure of the phosphoramidite derivative of a nucleotide is shown. It contains protective groups, namely, DMT (pink), benzoyl (blue), and diisopropyl amino and 2-cyanoethyl moieties (yellow). The next nucleotide-phosphoramidite is mixed with activating reagent such as 5-ethylthio-1H-tetrazole and introduced to the CPG for coupling. The activator renders the nucleotide-phosphoramidite reactive at the 5′ P-atom which leads to a phosphodiester bond with the last nucleotide on the CPG. Step 3: Unsuccessful coupling reactions are left with a reactive hydroxide group on the 5′ end of the CPG beads. These failed reactions are capped to replace the hydroxide with acetic anhydride using capping reagents such as 1-methylimidazzole in tetrahydrofuran/pyridine and tetrahydrofuran/acetic anhydride. Step 4: The oxidation state of the 5′ atom is changed from +3 to +5 using oxidizing reagent such as iodine in tetrahydrofuran/water/pyridine. The process is repeated until all nucleotides of a desired sequence are coupled from the 3′ end to the 5′ end. Below: Post-synthesis processing includes cleavage and release of the oligonucleotide from the CPG beads as well as hydrolysis of the bases and 5′ P atoms to remove the corresponding protective groups (shown in yellow and blue). Highly basic conditions such as ammonium hydroxide (7–30%) are typically used. The oligonucleotide can be used as is or further purified using reverse phase chromatography from any partial and incomplete sequences. Modified bases and dyes are inserted into the sequence using their phosphoramidite derivatives.