Novel reagents and procedures for immobilization of DNA on glass microchips for primer extension

A new method for attaching oligodeoxyribonucleotides to glass involving monoalkoxylated and dialkoxylated silanes and bromoacetamide/phosphorothioate linking chemistry has been developed. Three novel bromoacetamide silanes were synthesized for derivatization of glass microscope slides by traditional dipping methods. A thin film method that conserves silane and provides a consistent protocol for test experiments was also used. Oligonucleotides bearing 5'-phosphorothioates were synthesized by literature methods. Immobilization conditions were initially established by treatment of bromoacetamidosilyl slides with fluoresceinated oligonucleotides, which were imaged by confocal fluorescence microscopy. Spotting can be accomplished in water at oligonucleotide concentrations down to 0.1 mM. Oligonucleotides immobilized using this method can serve as primers for templated, polymerase-based extension reactions with a fluoresceinated dideoxynucleotide terminator. When such primers are formatted into small arrays, specific extension is observed only in the presence of complementary template, with the amount of immobilized primer reflected in the fluorescence signal.