RP-HPLC Method For The Purification Of Labeled Synthetic Oligonucleotides Using XTerra MS C₁₈ Columns

This application note demonstrates RP-HPLC method for the purification of labeled synthetic oligonucleotides using XTerra MS C₁₈ Columns.

Labeled oligonucleotide probes

Labeled oligonucleotides are utilized in quantitative polymerase chain reactions (qPCR), genotyping, DNA sequencing, and diagnostics. Among the most common labels are fluorescent dyes, various linkers, and biotin. Synthesis of labeled oligonucleotides is challenging, especially for multiple-labeled probes. The target product is typically contaminated with labeled and non-labeled failure products; both types of impurities, as well as the excess of uncoupled label, may compromise quality of the molecular biology assays.

XTerra Columns for enhanced oligonucleotide purification

Non-labeled oligonucleotides, labeled failure sequences, and excess of free label (dye) can be easily removed from the target product using XTerra HPLC Columns (Figure 1). XTerra MS C₁₈ Columns are packed with porous 2.5 μm, hybrid particles. XTerra hybrid particle technology has extended stability at temperatures and pH’s typically used for oligonucleotide separations (50–60 °C; pH 7-9). The column yields the high resolution efficiency required for successful oligonucleotide purification (See Lib. Ref #WA21955). The mobile phases are volatile and can be removed from the collected fractions by evaporation. The 4.6 x 50 mm column is suitable for purification of 50–200 nmole scale syntheses in a single injection. Larger amounts of oligonucleotides (200 nmole–1 μmole) can be purified using a 10 x 50 mm column or greater.

High purification yield and oligonucleotide purity

XTerra’s high performance allows for collection of the target fraction without sacrificing large amounts of the product (Table 1). When purifying 100 nmole with the 4.6 x 50 mm column, 75–80% of the target compound is typically recovered at a purity greater than 90%. Since RP-HPLC separates isomers of dye labels, we recommend this method for purification of single isomer dye-labeled oligonucleotides. The mobile phase components triethylammonium acetate (TEAA) and acetonitrile are volatile. Collected fractions are simply dried down and ready for use. Purification was monitored at two wavelengths: 260 nm (DNA detection, alternatively at 290 nm) and λmax of the label (dye labeled species only are detected). Real-time monitoring of two traces (chromatograms A & B, Figures 1 & 2) helps to identify a proper collection time window. Impurities eluting early during chromatography are the non-labeled failed products. The impurities eluting after the main peak are mostly dyelabeled failed sequences (Fountain et al., J. Chromatogr. B, 783 (2003) 61–72) (See Lib. Ref # WA20742).