DNA Oligonucleótides
General Characteristics.
Purification.
Following DNA synthesis, the completed DNA chain is released from the solid support by incubation in basic solutions such as ammonium hydroxide. This solution contains the required full-length oligo but also contains all of the DNA chains that were aborted during synthesis (failure sequences). If a 20-mer was synthesized, the solution would also contain 19 mer failures, 18 mer failures, 17 mer failures etc. The amount of failure sequences present is influenced by the coupling efficiency. These failure sequences can compete with the full-length product in some applications such as PCR, and may therefore need removing before the oligo can be used successfully.
Purification Method |
Description |
Benefit |
| Desalt | Oligos are processed through normal phase chromatography column which removes salts but not failure sequences. | A salt-free DNA solution, ready-to-use; suitable for many PCR and sequencing applications without further purification. |
| Cartridge | Based on reverse phase chromatography; removes failure sequences from the completed synthesis. | Provides full-length sequences needed in some applications |
| HPLC | Reverse Phase High Performance Liquid Chromatography (HPLC) removes failure sequences or unincorporated label the same way as cartridge purification. | Guarantees highly purified primer required in some applications (=>85% full-length). |
| PAGE | 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. |