Purification options

We make a number of purification options available depending on the needs of you particular molecular method. Choosing the appropriate purification option will ensure that your oligos are suitable for their application. Choosing just the right purification option will also ensure you have the best value in your oligos because increasing stringency of purification will by necessity diminish the final yield.   

Salt-free (“desalted”) oligos are suitable for applications such as microarrays, sequencing or qPCR primers. Reverse Phase Cartridge (RPC) purification is suitable to enrich the full-length product for oligos which contain 50 bases or less. For oligonucleotides containing modifications, such as fluorescent probes, we recommend more stringent purification such as single HPLC or dual HPLC. Single HPLC oligos are processed with either Reverse Phase HPLC (RP HPLC) or Anion Exchange HPLC (AX HPLC). Dual-HPLC oligos are processed with both RP HPLC and AX HPLC. Final QC specifications for custom-synthesized oligos are such that the observed mass must be within ±0.1% difference from the theoretical mass, as determined by electrospray mass spectrometry.

The following purification options are routinely available:

Salt-free (“desalted”)
All salt-free (“desalted”) oligos have been treated to remove by-products from the synthesis, cleavage, and deprotection methods. Further processing, such as Cartridge or HPLC Purification, may be recommended to increase overall purity of oligos used for sensitive applications. Please refer to the purification options below for more information and suggestions.

Reverse Phase Cartridge (RPC)
For unlabeled oligonucleotides (primers) Biosearch Technologies recommends RPC Purification which typically provides 70% purity*. Contaminants such as truncated sequences and other 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 of impurities by the binding affinity of the hydrophobic DMT group to the resin in the cartridge, while truncated sequences missing their DMT are washed away. The purified oligo is then eluted from the cartridge and the DMT removed before QC.

Reverse Phase HPLC (RP HPLC)
Reverse Phase HPLC is used to eliminate any fluorescent contamination remaining from probe synthesis. This impurity would otherwise elevate the background fluorescence and obscure detection of the signal. RP HPLC typically yields products with purity of ~80%*. The principle of this purification technique is similar to RPC, however the resins utilized provide greater sample capacity. Many of our probe types are well suited to RP HPLC purification including ValuProbe™ BHQ® Probes and BHQplus® probes.

Anion Exchange HPLC (AX-HPLC)
Anion Exchange HPLC is used to eliminate truncated oligos resulting from poor coupling during synthesis, such as quencher-only failure sequences that can afflict probe manufacture. When allowed to remain, this impurity competes with the probe to find the target sequence and may delay the cycles to threshold value in a qPCR assay.

Dual (AX +RP) HPLC
Dual-HPLC purification is Anion Exchange HPLC (AX HPLC) followed by Reverse Phase HPLC (RP HPLC) typically yields products with ~90%* purity and is the method of choice when more stringent purification processes are needed, such as with qPCR assays for industrial applications.

Other considerations

Oligos containing “wobbles”:
Oligos with degenerate residues (wobbles) require synthesizing two or more subspecies in unison. To avoid skewing the proportion of compounds, we elect to purify dual-labeled probes containing wobbles using only RP-HPLC rather than Dual HPLC (AX + RP). AX-HPLC can potentially enrich one compound over the other, whereas RP-HPLC uniformly enriches the desired subspecies. As such, RP HPLC represents our default purification for degenerate probes containing “wobble” bases.

Purification’s influence on final yield:
More stringent methods of purification will consequentially diminish the final yield of the delivered product. Salt-Free oligos therefore deliver the largest yields with the least % purity. Dual-HPLC delivers the highest purity but the smallest yield. Each application may tolerate a different purity profile. Our Technical Support team (techsupport@biosearchtech.com) is available for consultation, to assist in the selection of the appropriate purification method for your custom oligo needs.

Defining yield and purity specifications:
If you require a minimum nmol delivered, please specify the required yield with your order. We will suggest an appropriate synthesis scale to meet the required yield in accordance with the desired purification method. Similarly, we can also accommodate a minimum purity (e.g. 95%) that is elevated above our typical purity range. We are happy to discuss these custom specifications as long as the requirements are communicated in advance of the order.

*Final purity is determined by sequence, oligo length, selected modifications, and oligo type. Depending upon the complexity of the oligo, purity may vary from the levels as advertised.

Please Note: The purity percentages given are general guidelines and these values can vary depending on the sequence of the oligo.

What's next?  Learn about our available modifications or start building your custom oligonucleotide!