Coupling

FIGURE 21.14 Chemical Synthesis of DNA—Coupling

In order to couple a phosphoramidite nucleotide to the growing chain of DNA, the phosphoramidite moiety must be activated. Tetrazole activates the N of the diisopropylamino group by adding a proton. The diisopropylamino group is then displaced by the exposed 5'-OH of the acceptor nucleotide. The coupling reaction results in two nucleotides linked by a phosphite triester. Further reaction with iodine oxidizes this to a phosphate triester, which is then hydolyzed to a phosphodiester link by removal of the methyl group in the third position.

Blocking agents are used throughout the procedure to prevent the wrong groups from reacting.

The specific coupling of one nucleotide to another requires the addition of tetra-zole, which activates the phosphoramidite group on the new nucleotide. The phosphoramidite then forms a bond with the exposed 5'-hydroxyl group of the previous nucleotide (Fig. 21.14). Since this coupling reaction is not 100% efficient, any remaining unreacted 5'-hydroxyl groups must be blocked by acetylation before adding the next nucleotide. This is done using acetic anhydride plus dimethylaminopyridine. Unused but unblocked 5'-hydroxyl groups would react in the next synthetic cycle and give rise to incorrect DNA sequences. After coupling, nucleotides are linked by a relatively unstable phosphite triester (Fig. 21.14). This is oxidized by iodine to a phosphate triester, or phosphotriester. The methyl group that occupies the third posi-

tion of the phosphotriester is removed to give a phosphodiester linkage only after the whole DNA strand has been synthesized. After the phosphite oxidation step, the column is washed and treated with acid to expose the 5'-hydroxyl of the nucleotide that was just added. It is then ready for another nucleotide.

Since the amino groups of the bases are reactive they must also be protected throughout the whole reaction sequence. Benzoyl groups are normally added to protect the amino groups of adenine and cytosine, whereas, guanine is protected by isobutyryl group (thymine needs no protection as it has no free amino group). These groups are not shown in any of figures 21.11 through 21.14 and are only removed after synthesis of the whole strand of DNA. When all nucleotides have been added, the various protective groups are removed and the 5'-end of the DNA is phosphorylated, either chemically or by ATP plus polynucleotide kinase. After detaching from the column, the final product is purified by HPLC or gel electrophoresis to separate it from the defective shorter molecules that are due to imperfect coupling.

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