HYBRIDIZATION ASSAYS USING AN EXPRESSIBLE DNA FRAGMENT ENCODING FIREFLY LUCIFERASE AS A LABEL

We report the use of a new label, an expressible enzyme-coding DNA fra gment, for nucleic acid hybridization assays. The DNA label contains a firefly luciferase coding sequence downstream from a T7 RNA polymeras e promoter, The target DNA (200 bp) is denatured and hybridized simult aneously with two oligonucleotide probes. One of the probes is immobil ized in microtiter wells, via the digoxigenin/anti-digoxigenin interac tion, and the other probe is biotinylated. After completion of the hyb ridization, the hybrids are reacted with a streptavidin-luciferase DNA complex. Subsequently, the solid-phase bound DNA is expressed by coup led transcription/translation. The synthesized luciferase catalyzes th e luminescent reaction of luciferin with O-2 and ATP. The luminescence is linearly related to the amount of target DNA in the range of 5-500 0 amol. The CVs obtained for 20 and 100 amol of target are 6.5% and 10 .8%, respectively (n = 4).