Detection of single base substitutions in polynucleotides by capture with immobilized oligonucleotides.

Abstract We have improved a sandwich hybridization assay to detect single base substitutions in polymerase chain reaction (PCR) amplified DNA sequences. The target DNA was captured by an immobilized oligonucleotide and revealed using a second oligonucleotide coupled to an enzyme. Short oligonucleotides (13, 15 bases) were used to obtain specific hybridization at 37°C. We developed two different assay formats for rapid identification of PCR products: a microtitration plate format with oligonucleotides bound to polystyrene and a channelling assay using oligonucleotides immobilized on Sepharose, which did not require any separation step. The specificity and advantages of both methods are described.