Sandwich-type deoxyribonucleic acid hybridization assays based on enzyme amplified time-resolved fluorometry.
We report microtiter well-based sandwich-type DNA hybridization assays using enzyme amplified time-resolved fluorometry of Tb3+ chelates. The target DNA was hybridized with two adjacent and non-overlapping oligonucleotide probes, one oligonucleotide serving as the capture probe and the other as the detection probe. Two ligand-specific binding protein pairs were used alternately for capture of the hybrids to the solid phase and detection; the biotin-streptavidin and the digoxigenin-anti-digoxigenin interaction. In both cases, alkaline phosphatase was used as a reporter molecule and diflunisal phosphate as a substrate. The catalytic hydrolysis of the substrate produces diflunisal which forms ternary fluorescent complex with Tb(3+)-EDTA. Furthermore, we studied the effect of the probe labeling method and the position of the label on the sensitivity of the assays. The data suggest that capture of the hybrids through biotin-streptavidin and detection via digoxigenin-anti-digoxigenin offer 2-3 times higher sensitivity than the reverse configuration. The highest sensitivity was achieved with enzymatic labeling of capture and detection probes at the 3' end. A signal-to-background ratio of 4 was achieved for 0.2 fmol of target DNA. The RSD were better than 4%.
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
N H
NH
Chiu
T K
TK
Christopoulos
J
J
Peltier
D002460:Cell Line; D004247:DNA / Q000032:analysis*; D005470:Fluorometry; D006801:Humans; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity
D015345:Oligonucleotide Probes; D004247:DNA