In vitro Oligonucleotide Synthesis:
Oligonucleotides are synthesized according to manufacturer's procedures on either the Beckman Oligo 1000 or the ABI 392 synthesizer using the phosphoramidite chemistry . The desired oligonucleotide sequence is entered into the CPU affixed to the respective synthesizer, the reagent bottles are attached, and the column is inserted which contains the respective 3' nucleotide base-specific linked by the 3-OH group to the solid support, controlled-pore-glass (CPG) silica beads . The 5'-OH group of the base is blocked with a dimethyloxytrityl (DMT) group.
Oligonucleotide synthesis is carried out by a stepwise addition of nucleotide residues to the 5'-terminus of the growing chain until the desired sequence is assembled. Each addition is referred to as a synthetic cycle and consists of four chemical reactions:
Step 1: De-blocking (detritylation): The DMT group is removed with a solution of an acid, such as TCA or Dichloroacetic acid (DCA), in an inert solvent (dichloromethane or toluene) and washed out, resulting in a free 5' hydroxyl group on the first base.
Step 2: Coupling: A nucleoside phosphoramidite (or a mixture of several phosphoramidites) is activated by an acidic azole catalyst, tetrazole, 2-ethylthiotetrazole,
2-bezylthiotetrazole, 4,5-dicyanoimidazole, or a number of similar compounds. This mixture is brought in contact with the starting solid support (first coupling) or oligonucleotide precursor (following couplings) whose 5'-hydroxy group reacts with the activated phosphoramidite moiety of the incoming nucleoside phosphoramidite to form a phosphite triester linkage. This reaction is very rapid and requires, on small scale, about 20 s for its completion. The phosphoramidite coupling is also highly sensitive to the presence of water and is commonly carried out in anhydrous acetonitrile. Unbound reagents and by-products are removed by washing.
Step 3: Capping: After the completion of the coupling reaction, a small percentage of the solid support-bound 5'-OH groups (0.1 to 1%) remain unreacted and need to be permanently blocked from further chain elongation to prevent the formation of oligonucleotides with an internal base deletion commonly referred to as (n-1) shortmers. This is done by acetylating of the unreacted 5'-hydroxy groups using a mixture of acetic anhydride and 1-methylimidazole as a catalyst. Excess reagents are removed by washing.
Step 4: Oxidation: The newly formed tricoordinated phosphite triester linkage is not natural and is of limited stability under the conditions of oligonucleotide synthesis. The treatment of the support-bound material with iodine and water in the presence of a weak base (pyridine, lutidine, or collidine) oxidizes the phosphite triester into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleosidic linkage. This step can be substituted with a sulfurization step to obtain oligonucleotide phosphorothioates (see below). In the latter case, the sulfurization step is carried out prior to capping.
Courtesy :Wikipedia
