CAPILLARY AFFINITY GEL-ELECTROPHORESIS FOR COMBINED SIZE-DEPENDENT AND SEQUENCE-DEPENDENT SEPARATION OF OLIGONUCLEOTIDES

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 sel ectivity of bioaffinity recognition and high-resolution power of capil lary gel electrophoresis allows the on-line sequence- and size-specifi c separation of oligonucleotides, Both rigid gel formulations and visc ous replaceable polymer solutions having user-defined, single-stranded oligonucleotides covalently attached as recognition sequences are use d, Contrary to most known affinity systems in capillary electrophoresi s, which operate in a continuous mode, binding and release are accompl ished in two steps, effectively separating the affinity from the separ ation step, At low temperature, oligonucleotides with complementary se quences in the analyte solution will bind to the immobilized recogniti on sequence while unrelated oligonucleotides will continue to migrate. This step is a preseparation, removing all nonspecific solutes from t he sample, The release of the bound solutes is achieved at elevated te mperature, allowing a probe of cross-reactivity for a given biorecogni tion element. Applications for high-resolution separations of short ol igonucleotides and their mismatches are shown, and the potential for o n-line preconcentration and separation of dilute analyte solutions, th us effectively enhancing the sensitivity, is demonstrated.