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Chemical DNA Synthesis by Phosphoromidite Method
The phosphoroamidite method of chemical DNA synthesis is widely used. Nucleotides that are going to be used in the synthesis are normally modified or blocked at various sites in the molecule so that the side reactions are prevented during chain growth.

The amino groups in the adenine are derivatized by using benzoyl, whereas guanine and cytosine are derivatized by using isobutyryl and benzoyl groups respectively. Thymine is not treated because it does not have an amine group. The 3' -OH of the nucleotides are blocked by using diisopropylamine group.

Diisopropylomine group has a methyl group at its 3' ­phosphite group. 5 ' -PO 4 of the nucleotide is blocked by using dimethoxytrityl (DMT) group. Every possible site of non specific cross reaction is blocked before starting the extension or synthesis.

This structure where a nucleotide is blocked by DMT (at 5’-end) and diisopropylamine group with methyl group is called as phosphoroamidite.

The synthesis cycle of DNA starts with the attaching of the first nucleoside to the inert solid support (which is a controlled pore glass bead in most of the cases). The nucleoside is attached to the spacer by using 3' -OH of the nucleoside. After the attachment, the column is washed with acetonitrile to remove water and unbounded nucleotide.

Later the column is washed or flushed with argon to remove the acetonitrile. The TCA (trichloroacetic acid) is added into the column to remove DMT from the nucleotide attached to the spacer. The column is once again washed with acetonitrile to remove TCA and with argon to remove acetonitrile. This step is called as detritylation.

The next nucleotide in the form of phosphoroamidite is added along with tetrazole simultaneously. Tetrazole activates the phosphoroamidite so that its 3' -phosphite forms a covalent bond with the 5 ' 0 H group of the initial nucleoside. A covalent bond that is formed is in the form of a phosphate triester bond.

The column is flushed with argon to remove unincorporated phosphoroamidite and tetrazole. As phosphoroamidite does not bind all the support bound nucleosides, during the first coupling reactions, the unlinked residues must be prevented from linking to the next nucleotide during the following cycles.

Hence, the column is washed with acetic anhydride, and dimethyl aminopyridine is added to acetylate the unreacted 5 ' -OH groups. This step of coupling is required to prevent the synthesis of fragments with different lengths and sequences.

In the next stage, the column is flushed or washed with iodine mixture (KI + 12) for oxidation of the phosphate triester to form pentavalent phosphate triester. This step is required because phosphite triester bond is unstable and is most likely to break in the presence of either acid or base. This step is called as oxidation.

The process or cycle of detritylation, phosphoroamidite activation, coupling, capping and oxidation are repeated until the last nucleotide is added to the growing chain. The newly synthesized DNA are bound to the solid inert support with methyl group on the pentavalent phosphate triester bounded amino group.

The last nucleotide that is added contains a DMT at 5' end, which is removed by TCA. Hence the last nucleotide does not have 5' -PO4. From the DNA molecule, benzoyl and isobutyryl are removed and DNA nucleotide is detritylated.

Before the detritylation, a phosphate group is added at the 5' end with the help of alkaline phosphatase and A TP. Then the DNA molecule is cleaved from the spacer molecule leaving a 3' -OH terminus. From the column the DNA is eluted and purified by using either reverse phase high pressure liquid chromatography or by gel electrophoresis.
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