The DNA syntheisis cycle begins with the 3'-hydroxyl nucleoside
attached to a solid Controlled-Pore Glass (CPG) support through a
long spacer arm. This support allows excess reagents to be removed by
filtration and eliminates the need for purification steps between
base additions.
The first step in the synthesis is the removal of the dimethoxytrityl
(DMT) group with TCA to free the 5'-hydroxyl for the coupling
reaction. The next step, activation, is achieved by simultaneously
adding a phosphoramidite derivative of the next nucleotide and
tetrazole, a weak acid to the reaction chamber. The tetrazole
protonates the nitrogen of the phosphoramidite, making it susceptible
to nucliophilic attack. This intermediate is very reactive and the
following coupling step is complete in less than 30 seconds. The
5'-OH group of the phosphoramidite is blocked with the DMT group.
The capping step terminates any chains that did not undergo coupling.
The unreacted chain has a free 5'-OH which can be terminated or
capped by acetylation to become "failure products". This is achieved
with acetic anhydride and 1-methylimidazole. The DMT group of the
successful coupling step protects the 5'-OH end from being capped.
Although this step is not absolutely necessary for DNA synthesis, it
minimizes the length of impurities and thus facilitates trityl-on
HPLC Purification.
The internucleotide linkage is then converted from the less stable
phosphite to the phosphotriester in the oxidation step. Iodine is
used as the oxidizing agent and water as the oxygen donor. (For
phosphorothioate oligonucleotides, the internucleotide phosphite is
sulfurized between coupling and capping).
After oxidation, the DMT group is removed with trichloroacetic acid
and the cycle is repeated until chain elongation is complete. The
oligo is then cleaved from the solid support with concentrated
ammonium hydroxide. Ammonia treatment also removes the cyanoethyl
phosphate protecting groups. The crude DNA in ammonium hydroxide
solution is then heated at 65 degrees for 1 hour to remove the
protecting groups on the exocyclic amines of the base.
This product is lyophilized and can be used as is or purified by
reverse-phase HPLC.
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