How are oligonucleotides synthesized?
Oligonucleotides are produced using an automated DNA synthesizer that adds a base in the 3 to 5 direction. The addition of each base is termed a cycle. There are 4 basic steps to a cycle. 1.1 A new base is added to the 5 end of the growing chain. Approximately 99% of the available sites will react. 1.2 The sites that did not react are chemically capped. These capped bases will no longer be used in the synthesis and result in failed sequences. 1.3 The new base-to-base bond is stabilized by oxidation. 1.4 The 5 base has its protecting groups removed and is now ready to bond with the next added base when the first step is repeated. When the oligonucleotide synthesis has completed the oligo is cleaved form its solid support, which was used to hold it in place during synthesis. Protecting groups are then removed using concentrated ammonium hydroxide. While the oligo is ready for use in some application at this point, the commercial oligo vendors used by the DNA Facility will also remove the
