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Hybridization assays using as a label an expressible DNA fragment encoding firefly luciferase.

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Norman H. Chiu, Assistant Professor (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/

Abstract: We report the use of a new label, an expressible enzymecoding 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).

Additional Information

Publication
Language: English
Date: 1996
Keywords
chemistry, biochemistry, DNA, enzyme-coding, DNA label, hybridization, firefly luciferase