What different oligonucleotide purification options does Biosearch offer?

For unlabeled oligonucleotides (primers) Biosearch recommends Reverse Phase Cartridge (RPC) Purification which typically provides 70-75% purity. Contaminants such as truncated sequences, ammonium salts and impurities are removed from the final product. For oligos purified by RPC, the oligos are synthesized with the DMT group left on the final base which allows for separation by affinity of the DMT group to the resin in the cartridge. The principle behind this is that truncated sequences will not have the final DMT group and will not bind to the cartridge and will be washed away. The purified oligo is then eluted from the cartridge. For oligos 50 bases or less the RPC provides high enriched full length product.

For dual-labeled probes, we recommend either the single HPLC or dual HPLC. Single HPLC oligos are processed with Reverse phase HPLC. Dual HPLC oligos are processed with both Reverse Phase and Anion Exchange HPLC.

Reverse Phase HPLC is well-suited to eliminate any fluorescent contamination remaining from the probe synthesis. When left unremoved, this impurity elevates the baseline fluorescence and obscures the detection of probe cleavage. RP-HPLC typically yields purity of products around 70-90% along with the highest yields. The purification technique is similar to Reverse Phase Cartridge but the resins provide greater sample capacity.

Anion Exchange is well-suited to eliminate quencher-only failure sequences resulting from poor reporter or base coupling during the synthesis. When left unremoved, this impurity competes with the probe to find the amplicon and can slightly delay the cycle threshold value.

Dual-HPLC (Reverse Phase and Anion Exchange HPLC) typically results in products with 85-90+% purity.

View our Default and Recommended Methods of Purification Chart for more details.

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