Quality Control of DNA Synthesis using Fast Ion-pair Reversed-phase High Performance Liquid Chromatography

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Kenneth J. Fountain, Martin Gilar, John C. Gebler [Waters]
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Current methods of DNA synthesis can routinely produce high quality oligonucleotides, provided that automated synthesizers are in optimal operating condition. While suboptimal synthesis can still yield acceptable and usable oligonucleotides, diagnostic and therapeutic applications require high purity probes. A method utilizing ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC) with ultraviolet (UV) detection has been developed for the routine quality control (QC) of synthetic oligonucleotides up to 30mer in length. Separation was performed on a 4.6 20-mm MS C18, 2.5 mm column. Mobile phases were comprised of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and triethylamine (TEA) with a concave methanol (MeOH) gradient. Resolution of the target product from N – 1 impurities was routinely achieved for all DNA fragments analyzed, including oligonucleotides with mixed base sequences (up to 30mer). The 5-minute per sample duty cycle allows for fast diagnosis of the performance of DNA synthesizers.

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