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.