Composition dependent separation of oligonucleotides by capillary electrophoresis in acidic buffers with application to the quality control of synthetic oligonucleotides

Abstract

Oligonucleotides have become a widely used tool in molecular biology and molecular diagnostics. Their parallel synthesis in large numbers has raised the request for fast and informative analytical tools for their quality control. Here we propose an alternative to current analytical methodologies based on capillary electrophoresis at very low pH in free solution. In these non-classical analytical conditions oligonucleotides can be discriminated for their base composition, thus adding a further dimension to the classical electrophoretic sizing performed in sieving media at moderately basic pH. We have tested several separating conditions at various pH values on a very large set of samples (about 200 synthetic oligonucleotides) ranging from 10 to 50 residues and with different terminal modification including amine, phosphate, biotin, fluorescein and other fluorescent dyes. We have been able to characterize the quality of these synthetic products and to detect base redundancies (i.e. the copresence of different bases in a single position of the oligonucleotide chain) in oligonucleotides up to 50 bases long, thus posing the basis for the application of this method to the emerging field of detection mutation in complex genomes analysis.