High-throughput screening method for promoter activity using bead display and a ligase ribozyme

Abstract

In this report, we describe the development of a novel in vitro high-throughput system for detecting and screening promoter activity; the method employs emulsified reactions and a ligase ribozyme. In our study, a promoter DNA fragment containing the ribozyme gene was immobilized on a bead by using emulsion PCR, followed by in vitro transcription of the immobilized DNA in water-in-oil emulsions. Owing to the self-ligation activity of the ribozyme, it was co-transcriptionally linked to the active promoter immobilized on the beads. The bead complex containing the active promoter sequence was then labeled by reverse transcription with a fluorescently labeled primer. Employing flow cytometry, the fluorescence intensity corresponding to the strength of each promoter was observed, indicating the applicability of the system for promoter evaluation. Moreover, two rounds of screening with T7 RNA polymerase using a cell sorter enriched the T7 promoter fragment by 70 folds from a 1:100 mixture of T7 promoter and SP6 promoter fragments, suggesting that this system can be used to screen promoters.