Novel hybridization assay configurations based on in vitro expression of DNA reporter molecules.

OBJECTIVES

To develop and study novel microtiter well based DNA hybridization assays in which the DNA serves as a reporter molecule.

METHODS

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.

RESULTS

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%.

CONCLUSION

The expression yield is significantly improved with the separate transcription/translation protocol. Both assay configurations offer high sensitivity and are easily automatable.