Laios E, Ioannou P C, Christopoulos T K
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Clin Biochem. 1998 Apr;31(3):151-8. doi: 10.1016/s0009-9120(98)00006-x.
To develop and study novel microtiter well based DNA hybridization assays in which the DNA serves as a reporter molecule.
Two hybridization assay configurations are proposed. In configuration A the target DNA is end-labeled with biotin and captured to streptavidin-coated wells. The one strand is removed by NaOH treatment and the other is hybridized with a dATP-tailed oligonucleotide probe. Configuration B involves simultaneous hybridization of heat-denatured target DNA with a biotinylated capture probe (immobilized on streptavidin-coated wells) and a dATP-tailed detection probe. In both configurations the hybrids are reacted with dTTP-tailed luciferase-coding DNA fragment followed by in vitro expression of the DNA on the solid phase. This is accomplished either by a coupled transcription/translation or by sequential transcription and translation reactions optimized separately.
The signal-to-background ratios for configuration A at 0.93 fmoles target DNA were 2.6 and 16.7 with the coupled and the separated transcription/translation protocols, respectively. As low as 0.1 fmoles target DNA can be detected with the separate transcription/translation protocol with a signal-to-background ratio of 2.7. The signal-to-background ratios obtained for 0.1 fmoles target DNA with configuration B using the coupled and the separate expression protocols were 2.2 and 4.6, respectively. The average CV was 10%.
The expression yield is significantly improved with the separate transcription/translation protocol. Both assay configurations offer high sensitivity and are easily automatable.
