What is solid-phase oligonucleotide Synthesis
Oligonucleotides are produced using solid-phase synthesis chemistry. Solid-phase synthesis is carried out on a solid support held between filters, in columns that enable all reagents and solvents to pass through freely using an automated DNA/RNA synthesizer that adds a base in the 3' to 5' or 5' to 3' direction. The addition of each base is called a cycle. There are 4 basic steps to a cycle. A new base is added to the 5' end of the growing chain. Approximately 99% of the available sites will react. The unreactive sites are chemically capped. These capped bases will no longer be used in the synthesis and result in truncated sequences. The growing base-to-base bond is stabilized by oxidation. 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. Oligonucleotide is further desalted to remove the reaction salts and further purified by HPLC or PAGE. Solid-phase oligonucleotide synthesis methods can drive reactions quickly to completion using large excesses of solution-phase reagents. The impurities and excess reagents are washed away and no purification is required after each synthesis cycle. This method is amiable to automation on synthesizers.
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08/20/2013