Abstract
Purification of oligonucleotides has traditionally relied on mobility-based separation methods. However, these are imperfect, biased, and difficult to scale high multiplex. Here, we present a method for simultaneous purification of many oligonucleotides that also normalizes concentrations. The method uses a rationally designed randomer capture probe to enrich for oligos with perfect 5′ sequences, based on the observation that synthesis errors are correlated: product molecules with one or more deletions in one region are also more likely to have deletions in other regions. Next-generation sequencing analysis of 64-plex 70 nt purification products show a median 78% purity, a significant improvement over polyacrylamide gel electrophoresis and high pressure liquid chromatography (60% median purity). Additionally, 89% of the oligo products are within a factor of 2 of the median concentration.
Highlights
Purification of oligonucleotides has traditionally relied on mobility-based separation methods
For many applications where high purity oligos are desired, high pressure liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) post-synthesis purification is used to remove the majority of synthesis failures
Alternative purification technologies based on polymerization[20], mismatch recognition enzymes[21,22,23], or phase tags[24] have been proposed and demonstrated for simultaneous purification of pools of oligos, but these generally do not show improved purity over HPLC/PAGE, and the product sequences have not been systematically analyzed at the single-molecule level to characterize true purity
Summary
Purification of oligonucleotides has traditionally relied on mobility-based separation methods. Application of HPLC or PAGE to a pool of oligos does not achieved the desired purification effect, because in the vast majority of use cases the oligos in the pool will have different mobilities due to length and sequence variations. In addition to improving oligo purity, the SNOP method normalizes the concentrations of the oligo products, so that all products are at similar concentrations in the final pool This feature of SNOP allows the user to overcome synthesis yield variations, and prevents a few oligo species from dominating a pool. In this manuscript, we experimentally demonstrate 64-plex and 256-plex SNOP, and characterized the product purities using next-generation sequencing (NGS). The high purity of SNOP products, combined with the highthroughput nature of SNOP, renders SNOP an attractive and affordable method for modern research and development needs
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