Automated synthesis of oligonucleotides is carried out from the 3'- to the 5'-terminus. During the synthesis process, failure sequences (caused by incomplete monomer addition) are end-capped. Assuming that end-capping is efficient, only the full-length sequence should contain a dimethoxytrityl (DMT) group at the 5'-terminus on completion of the synthesis. Since the DMT group binds strongly to reverse phase supports, the full-length sequences can be retained in a cartridge while the failure sequences are eluted. The DMT group is then removed on the cartridge and the purified product is eluted.
Deprotection of synthetic oligonucleotides with ammonium hydroxide (30%; SG=0.88) removes the sequence from the support, while removing all base-labile protecting groups. Following this procedure, there is no need to lyophilize the ammonium hydroxide solution since the polymeric resin is stable at high pH. However, if the product has been lyophilized, simply reconstitute it in 0.1M triethylamine acetate (TEAA).
As with Poly-PakTM cartridges, Glen-PakTM cartridges utilize the 5’ DMT retained on an oligonucleotide to specifically bind full-length sequences to the support. During the purification process, the failure sequences are eliminated and the DMT is subsequently removed, allowing for elution of purified product.
The principle of the Glen Research gel filtration column, Glen Gel-Pak™ , is based on size exclusion chromatography that separates molecules according to the hydrodynamic volume of the molecule in aqueous solutions.
Glen-Pak™ DNA and RNA cartridges have advantages over Poly-Pak cartridges in that a single loading of the diluted crude deprotection solution is all that is necessary.
Oligo-affinity supports (OAS) should ideally be compatible with automated synthesis, should be non-friable, should not shrink or swell, and should have low non-specific binding of the proteins or DNA.
Since the DMT group binds strongly to reverse phase supports, the full-length sequences can be retained in a cartridge while the failure sequences are eluted.