Oligonucleotide synthesis has revolutionized biochemical and lifescience research on many levels. The last few decades, chemical synthesis of oligonucleotides is universally performed making use of phosphoramidite chemistry (1). Although new methods have been explored and show promise for the future, there is currently no other viable method for high-speed and high throughput synthesis of custom oligonucleotides (2). In general, the oligonucleotide synthesis process can be summarized as follows:
- Determination of the synthesis strategy and selection of the optimal reagents (synthesis strategy).
- Subsequent addition of nucleotide residues and, optionally, modifications. (synthesis cycle, coupling efficiency).
- Separation of the oligonucleotide from its starting material and removal of protecting groups that prevented side-reactions during synthesis. (resin cleavage and oligo deprotection).
- Purification of the oligonucleotide from (post)synthesis reaction residues and optionally further purification steps (purifications).
- Quantification, Quality Control, transfer to final format and order shipment (quality control).
- Analysis of data on quality parameters and functionality of the oligo in its application to serve as input for maintaining and optimizing the complete production process to the quality standards requested by the market (data analysis).
(1) Synthesis of DNA/RNA and Their Analogs via Phosphoramidite and H-Phosphonate Chemistries doi.org/10.3390/molecules181114268
(2) Technological challenges and milestones for writing genomes doi: 10.1126/science.aay0339