Q14. What is coupling efficiency?

Oligos are made in small columns filled with a solid support, usually glass or polystyrene beads, which are designed to anchor the growing DNA chain. The DNA synthesis consists of a series of chemical reactions: 1. Deblocking: The first base from 3´ end is via a chemical linker attached to the solid support. The trityl protecting group on 5’ OH is removed with an acid to produce a free 5´ OH ready for reaction with the next base. 2. Coupling: The next base is added and coupled to the first base. 3. Capping: 1-2% of the free 5´OH is not reacted with the next base and is then capped with acetic anhydride. These failed bases will play no further part in the synthesis cycle. 4. Oxidation: The established phosphite group between the first and the second base is oxidized with iodine to phosphate. 5. Deblocking: The trityl group on 5´-OH on the second base is removed to produce a free 5´ OH ready for reaction with the next base. Each cycle of reactions results in the addition of a single base. In this way a chain of bases can be connected by repeating the synthesis cycles till the desired length is achieved. A computer controls the reagent delivery system. The yield of each step is called the coupling efficiency. The coupling efficiency is around 99%. The table shows the effect of a difference in coupling efficiency when an oligo is synthesized.
% yield of a n-mer 99.5% Coupling 99% Coupling 98% Coupling
20 bases 90 82 67
40 bases 82 67 45
60 bases 74 55 30
80 bases 67 45 20
100 bases 61 37 14

 



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