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