FAQ

Biosearch Technologies offers a full range of purification options including: Salt-free, Reverse Phase Cartridge (RPC), Reverse Phase HPLC (RP-HPLC), Anion Exchange HPLC (AX-HPLC) and Dual-HPLC (AX-HPLC followed by RP-HPLC). They are listed from least to most stringent, with the appropriate purification depending entirely on the application.

For unlabelled oligonucleotides, such as qPCR primers, Salt-free or RPC purification is appropriate. For other applications using unmodified oligonucleotides we encourage RPC purification which typically provides ~70 % purity. With RPC purification, contaminants such as truncated sequences, ammonium salts and impurities are removed from the final product. In this process, 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. Truncated sequences will not have the final DMT group, will not bind to the cartridge and will be washed away.

RP-HPLC is selected to eliminate fluorescent contaminants that remain following synthesis of a labelled oligo. When allowed to persist, this impurity elevates the baseline fluorescence and obscures the detection of probe signal. RP-HPLC typically yields products with ~80 % purity. This purification technique is similar to RPC purification except the resins provide greater sample capacity.

AX-HPLC is selected to eliminate failure sequences that result from poor reporter or base coupling during the synthesis. When allowed to persist, this impurity competes with the oligo for binding to the target sequence which may result in delayed CT values in a qPCR reaction.

For BHQ™ Probes we recommend at a minimum RP-HPLC purification, but default to Dual-HPLC which typically provides products with ~90 % purity.

In oligonucleotides containing wobbles, we avoid AX-HPLC which skews the ratio of different species synthesized in unison.

For more information, please review our Default and Recommended Methods of Purification Chart.