COMPARISON OF AN INTERCALATING DYE AND AN INTERCALANT-ENZYME CONJUGATE FOR DNA DETECTION IN A MICROTITER-BASED ASSAY

Two methods have been developed for the detection of DNA immobilized o n the surface of microtiter wells. An intercalating dye, 3,6-diaminoac ridine, is used in stain and rinse solutions, so that measured absorba nce values (450 mn) reflect the sum of DNA-bound and free dye, With di aminoacridine, signal increases of 0.056 +/- 0.010 were achieved on im mobilizing double-stranded calf thymus DNA. An intercalant-enzyme conj ugate, consisting of an average of four daunomycin moieties covalently bound to each glucose oxidase, was shown to provide a 10 fold signal enhancement (optimum 0.25 mu M, with rinsing and peroxidases-dianisidi ne detection) compared to diaminoacridine, due to catalytic amplificat ion; signals of 0.50 +/- 0.05 were obtained. This conjugate possesses 56% of the activity of native glucose oxidase and was prepared using w ater-soluble carbodiimide and N-hydroxysuccinimide reagents. Single-st randed DNA was immobilized onto avidin-coated polystyrene plates and c ommercially available (Covalink) plates possessing secondary amine gro ups. Following hybridization with complementary DNA, detection was per formed with the daunomycin-glucose oxidase conjugate. Both immobilizat ion methods showed optimum DNA concentrations of 0.10 mu g/mL, and max imum signal intensities were obtained when >0.5 mu g/mL complementary DNA was present in the hybridization solution. Some nonspecific bindin g of the intercalant-enzyme conjugate was suggested by results obtaine d with avidin-coated polystyrene plates, but not with Covalink plates.