Purification Options

Unpurified, desalted oligos are suitable for applications such as microarrays, sequencing or qPCR. Labeled oligos require more stringent conditions and a higher level of purification. We also recommend that any oligo over 40 nucleotides receive further purification. Final Mass Spec QC specifications for all of our primer and probe oligos is ±0.1% difference from theoretical mass.

The following purification options are routinely available:

Reverse Phase Cartridge (RPC)
For unlabeled oligonucleotides (primers) Biosearch Technologies recommends Reverse Phase Cartridge (RPC) Purification which typically provides 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 highly enriched full-length product. For labeled oligonucleotides, such as fluorescent probes, we recommend either single HPLC or dual HPLC. Single HPLC oligos are processed with Reverse phase HPLC. Dual HPLC oligos are processed with both Reverse Phase (RP) and Anion Exchange (AX) HPLC.

Reverse Phase HPLC (RP-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 ~85%* along with the highest yields. The purification technique is similar to Reverse Phase Cartridge but the resins provide greater sample capacity. Our ValuProbe oligos are RP-HPLC purified.

Anion Exchance HPLC (AX-HPLC)
Anion Exchange HPLC 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 (AX/RP) HPLC
Anion Exchange HPLC followed by Reverse Phase HPLC typically results in products with ~90%* purity and is the method of choice when more stringent purification processes are needed for applications such as multiplexing reactions.

*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.

Default and Recommended Methods of Purification
Oligonucleotide Type	Purification	Typical Purity
Dual-labeled Probes
Small Scale Probes	Dual HPLC: AX/RP	85%
Small Scale Probes/ValuProbe	RP-HPLC	80%
Large Scale Probes	Dual HPLC: AX/RP	90%
CAL Fluor/BHQ Probes	Dual-HPLC	80%
Quasar 670/BHQ Probes	Dual-HPLC	90%
BHQplus Probes	RP-HPLC	70%

Single-labeled Probes
5' Labeled Probes	RP-HPLC	75%

Primers
Small Scale Primers	RPC	N/A
Large Scale Primers	RPC	70%
AX Purified Primers	AX-HPLC	90%

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

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Purification Options

Default and Recommended Methods of Purification