Purity
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.
Purification options
- 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) |
|---|---|---|---|---|
| 25 nmol | 2 | n/a | n/a | n/a |
| 50 nmol | 4 | 2 | 1 | 0.5 |
| 200 nmol | 16 | 10 | 3 | 1 |
| 1 μmol | 50 | 25 | 5 | 5 |
| 10 μmol | 500 | n/a | 15 | 50 |