Scale of Synthesis vs. Synthesis Yield
The scale of synthesis refers the amount of the first “base” used at the start of the DNA synthesis.
There are several steps to DNA synthesis, including:
- the step-wise coupling of each “base”
- the cleavage of the oligonucleotide from the solid support
- removal of various protecting groups
- desalting of the oligonucleotide
- any additional purification steps, if desired
Since none of these steps is 100% efficient, the actual yield of oligonucleotide will be less than the scale at which it was synthesized. Oligonucleotide synthesis starts at the 3′-terminal base, which is attached to the solid support. Bases are added one at a time in the 3′-end to 5′-end direction. For each coupling/capping reaction, the efficiency of the reaction is between 97.0 – 99.5% efficient; we observe an average coupling percentage of 98.5%.
For example, at the end of the synthesis of a 20-mer, which has undergone 19 coupling reactions, the amount of full-length material ranges between 0.9720 to 0.9920, or 54% to 91% (average = 74%). The remaining 9 – 46% of the material would consist of approximately equal amounts of the 19-mer, 18-mer, 17-mer, etc.
Following synthesis, the oligonucleotide is deprotected in concentrated ammonium hydroxide and desalted on a gel filtration column. If desired by the customer, additional HPLC or PAGE purification can also be performed. The desalting and additional purification steps are not 100% efficient. Depending on the length of the oligonucleotide, the complexity of the synthesis, and the type of purification, the yield from a 0.2 micromole scale synthesis can be as low as 10 nanomoles or greater than 100 nanomoles.