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 on the surface of microtiter wells. An intercalating dye, 3,6-diaminoacridine, is used in stain and rinse solutions, so that measured absorbance values (450 nm) reflect the sum of DNA-bound and free dye. With diaminoacridine, signal increases of 0.056 +/- 0.010 were achieved on immobilizing double-stranded calf thymus DNA. An intercalant-enzyme conjugate, 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 microM, with rinsing and peroxidase-o-dianisidine detection) compared to diaminoacridine, due to catalytic amplification; signals of 0.50 +/- 0.05 were obtained. This conjugate possesses 56% of the activity of native glucose oxidase and was prepared using water-soluble carbodiimide and N-hydroxysuccinimide reagents. Single-stranded DNA was immobilized onto avidin-coated polystyrene plates and commercially available (Covalink) plates possessing secondary amine groups. Following hybridization with complementary DNA, detection was performed with the daunomycin-glucose oxidase conjugate. Both immobilization methods showed optimum DNA concentrations of 0.10 microgram/mL, and maximum signal intensities were obtained when > 0.5 microgram/mL complementary DNA was present in the hybridization solution. Some nonspecific binding of the intercalant-enzyme conjugate was suggested by results obtained with avidin-coated polystyrene plates, but not with Covalink plates.
Kolakowski
B
B
Department of Chemistry and Biochemistry, Concordia University, Montreal, Canada.
Battaglini
F
F
Lee
Y S
YS
Klironomos
G
G
Mikkelsen
S R
SR
D000818:Animals; D002417:Cattle; D004247:DNA / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000032:analysis; D003630:Daunorubicin; D005949:Glucose Oxidase; D007364:Intercalating Agents; D008832:Microchemistry / Q000379:methods; D011370:Proflavine
D004277:DNA, Single-Stranded; D007364:Intercalating Agents; D004247:DNA; D011370:Proflavine; D005949:Glucose Oxidase; D003630:Daunorubicin