Reverse phase (RP) cartridges have been popular1 for some time for rapid and inexpensive purification of synthetic oligonucleotides. The use of a polystyrenedivinylbenzene copolymer packing in cartridges or columns overcomes several disadvantages usually associated with silica gel based RP cartridges. Polystyrene is stable in the pH range 1-13, thus the ammonia deprotection solution, diluted with water, may be loaded directly onto the packing. Also, after elution of failure sequences, the dimethoxytrityl group may be removed while the oligonucleotide is support-bound. The fully deprotected product can then be eluted and isolated by lyophilization. Polystyrene packing may also be used for desalting a normal or dye-labelled oligonucleotide eluted from a gel.
The packing Glen Research has selected for use in purification cartridges is Polymer RP-1, a highly purified polystyrene resin with a particle size distribution of 12-20 micron. Glen Research Poly-Pak™ cartridges, therefore, contain 125mg of Polymer RP-1 packing retained by frits in a packed chromatographic bed. The cartridges are molded from translucent polypropylene. The Poly-Pak cartridge housing has a female luer fitting at the inlet to attach a luer-hub syringe reservoir for fast, convenient sample processing. A male luer fitting at the outlet is used to direct flow to a collection tube or to attach the cartridge to a vacuum manifold for parallel processing of multiple samples.
Poly-Pak cartridges exhibit the following attractive features:
1 ) The ability to prepare at least 10 A260 units and as much as 30 A260 of purified oligonucleotide. This feature is highly significant for the production of oligonucleotides destined for use in DNA amplification experiments.
2) Wash volumes are diminished to a level where the total eluent is less than 15ml. This speeds the process dramatically in comparison to other reverse phase cartridges.
3) Product oligonucleotides are eluted in 4 drops of aqueous acetonitrile, thereby minimizing the time needed for solvent evaporation.
The ability of Poly-Pak cartridges to produce high quality oligonucleotides is demonstrated in the purification of a 24-mer, as shown in the Figures to the right. Figure 1 contains the RP HPLC trace of the crude DMT-ON oligonucleotide after deprotection with ammonia. In Figure 2, the purified DMT-ON 24-mer has been eluted from the cartridge to illustrate the purity increase achieved by the cartridge. In Figure 3, The DMT group has been removed and the fully deprotected 24-mer has been isolated in 4 drops of aqueous acetonitrile.