Oligonucleotide-Enzyme Conjugates

For nucleic acid hybridizations, the choice of nonisotopic label is constrained by hybridization and wash conditions, the sensitivity needed in the assay, and the detection method. Several recent reviews (1 –4 ) have summarized methods of labeling synthetic DNA probes. In spite of significant advances in the detection of fluorescent and luminescent labels, enzymes continue to be the most sensitive reporter groups. For most applications, direct enzyme labels offer the best overall performance, with highest sensitivity, least background, and rapid detection. Several enzymes, particularly alkaline phosphatase and horseradish peroxidase, are compatible with standard hybridization conditions, and allow detection by a number of endpoints, including soluble color, dye deposition, fluorescence, and luminescence. Reported methods (5 ) for the crosslinking of enzymes to cloned double-stranded DNA does not work acceptably for oligonucleotides, giving little or no hybridization.