Hybridization Assays Using an Expressible DNA Fragment Encoding Firefly Luciferase as a Label

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Abstract
We report the use of a new label, an expressible enzyme-coding DNA fragment, for nucleic acid hybridization assays. The DNA label contains a firefly luciferase coding sequence downstream from a T7 RNA polymerase promoter. The target DNA (200 bp) is denatured and hybridized simultaneously with two oligonucleotide probes. One of the probes is immobilized in microtiter wells, via the digoxigenin/anti-digoxigenin interaction, and the other probe is biotinylated. After completion of the hybridization, the hybrids are reacted with a streptavidin−luciferase DNA complex. Subsequently, the solid-phase bound DNA is expressed by coupled transcription/translation. The synthesized luciferase catalyzes the luminescent reaction of luciferin with O2 and ATP. The luminescence is linearly related to the amount of target DNA in the range of 5−5000 amol. The CVs obtained for 20 and 100 amol of target are 6.5% and 10.8%, respectively (n = 4).