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 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).
Chiu
N H
NH
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Christopoulos
T K
TK
D000818:Animals; D001483:Base Sequence; D001517:Coleoptera / Q000201:enzymology*; D004247:DNA; D008156:Luciferases / Q000235:genetics*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012588:Scintillation Counting; D013050:Spectrometry, Fluorescence; D013698:Templates, Genetic
D015345:Oligonucleotide Probes; D004247:DNA; D008156:Luciferases