NOVEL HYBRIDIZATION ASSAY CONFIGURATIONS BASED ON IN-VITRO EXPRESSIONOF DNA REPORTER MOLECULES

Objectives: To develop and study novel microtiter well based DNA hybri dization assays in which the DNA serves as a reporter molecule. Method s: Two hybridization assay configurations are proposed. In configurati on A the target DNA is end-labeled with biotin and captured to strepta vidin-coated wells. The one strand is removed by NaOH treatment and th e other is hybridized with a dATP-tailed oligonucleotide probe. Config uration B involves simultaneous hybridization of heat-denatured target DNA with a biotinylated capture probe (immobilized on streptavidin-co ated wells) and a dATP-tailed detection probe. In both configurations the hybrids are reacted with dTTP-tailed luciferase-coding DNA fragmen t followed by in vitro expression of the DNA on the solid phase. This is accomplished either by a coupled transcription/translation or by se quential 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 fmol es target DNA can be detected with the separate transcription/translat ion protocol with a signal-to-background ratio of 2.7. The signal-to-b ackground ratios obtained for 0.1 fmoles target DNA with configuration B using the coupled and the separate expression protocols were 2.2 an d 4.6, respectively. The average CV was 10%. Conclusion: The expressio n yield is significantly improved with the separate transcription/tran slation protocol. Both assay configurations offer high sensitivity and are easily automatable. Copyright (C) 1998 The Canadian Society of Cl inical Chemists.