Oligos are made using a DNA synthesizer, which is a computer-controlled reagent delivery system. DNA is synthesized in the 3’→5’direction, and each base addition is accomplished through a series of chemical reactions. Since no chemical reaction is 100% efficient, during DNA synthesis, the maximum coupling efficiency obtainable is normally around 99%. This means that every time a base is added, approximately 1% of available bases fail to react with the newly added base. The failure sequences inherent to the oligo synthesis process can compete with the full-length product in some applications and may therefore need removing through purification before the oligo can be used successfully.
- Desalting (available for oligos with 25nmol scale: 10-100 bases, or 50nmol and higher scales: 5-100 bases in length): Oligos are processed through normal phase chromatography columns, which remove salts but not failure sequences. The result is a salt-free, ready-to-use DNA solution, suitable for many PCR and sequencing applications without further purification.
- Cartridge (available for oligos with 50nmol – 1umol scale, 7-55 bases in length): Based on reversed-phase chromatography, removes failure sequences from the completed synthesis. Provides enrichment for full length sequences needed in some applications (≥75%).
- HPLC (available for oligos with 50nmol or higher scales, 10-55 bases in length): Reversed-phase high performance liquid chromatography (HPLC) removes failure sequences or unincorporated labels (e.g. fluorescent dye) Guarantees highly purified primer required in some applications (≥85%)
- PAGE (available for oligos with 50nmol or higher scales, 7-100 bases in length): Method used to differentiate full-length product from failure sequences based on size and conformation. Provides the highest percentage of full-length oligos (>85%) required for certain demanding applications such as mutagenesis or adapter production. This is the only purification method effective for oligos over 60 bases.
Purification (for >20 bases)
|Synthesis scale||Desalted (OD)||Cartridge (OD)||HPLC (OD)||PAGE (OD)|