Capillary Affinity Gel Electrophoresis for Combined Size- and Sequence-Dependent Separation of Oligonucleotides

Abstract

An interesting new approach to capillary affinity gel electrophoresis (CAGE) has been developed for the selective capture and separation of homopolymer and heteropolymer oligonucleotides. The combination of selectivity of bioaffinity recognition and high-resolution power of capillary gel electrophoresis allows the on-line sequence- and size-specific separation of oligonucleotides. Both rigid gel formulations and viscous replaceable polymer solutions having user-defined, single-stranded oligonucleotides covalently attached as recognition sequences are used. Contrary to most known affinity systems in capillary electrophoresis, which operate in a continuous mode, binding and release are accomplished in two steps, effectively separating the affinity from the separation step. At low temperature, oligonucleotides with complementary sequences in the analyte solution will bind to the immobilized recognition sequence while unrelated oligonucleotides will continue to migrate. This step is a preseparation, removing all nonspecific solutes from the sample. The release of the bound solutes is achieved at elevated temperature, allowing a probe of cross-reactivity for a given biorecognition element. Applications for high-resolution separations of short oligonucleotides and their mismatches are shown, and the potential for on-line preconcentration and separation of dilute analyte solutions, thus effectively enhancing the sensitivity, is demonstrated.