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Related Experiment Video

Updated: May 20, 2026

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
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High-throughput screening method for promoter activity using bead display and a ligase ribozyme.

Takaaki Kojima1, Shoji Ohuchi, Yurie Ito

  • 1Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.

Journal of Bioscience and Bioengineering
|July 28, 2012
PubMed
Summary

We developed a novel in vitro system using emulsified reactions and a ligase ribozyme to detect and screen promoter activity. This high-throughput method enables efficient promoter evaluation and enrichment for specific promoter sequences.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Biochemistry

Background:

  • Assessing promoter activity is crucial for gene expression studies.
  • Existing methods for promoter screening can be low-throughput or complex.
  • A need exists for efficient, high-throughput systems to evaluate promoter strength and identify optimal sequences.

Purpose of the Study:

  • To develop and validate a novel in vitro high-throughput system for detecting and screening promoter activity.
  • To demonstrate the system's capability for promoter evaluation and enrichment.
  • To utilize emulsified reactions and a ligase ribozyme for promoter analysis.

Main Methods:

  • Immobilization of promoter DNA fragments containing a ribozyme gene onto beads via emulsion PCR.
  • In vitro transcription within water-in-oil emulsions, leading to co-transcriptional linking of the ribozyme to the promoter.

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  • Fluorescent labeling of bead-bound promoter complexes via reverse transcription.
  • Analysis of fluorescence intensity using flow cytometry for promoter strength evaluation.
  • Enrichment of specific promoter fragments using a cell sorter in a high-throughput screening process.

    • Main Results:

    • Successfully developed a novel in vitro high-throughput system for promoter activity detection.
    • Demonstrated the system's applicability for promoter evaluation through flow cytometry analysis of fluorescence intensity.
    • Achieved a 70-fold enrichment of T7 promoter fragments from a mixed population using two rounds of screening with T7 RNA polymerase and a cell sorter.
    • Confirmed the co-transcriptional linking of the ribozyme to the immobilized active promoter.

    Conclusions:

    • The developed in vitro system is effective for high-throughput detection and screening of promoter activity.
    • The system allows for quantitative evaluation of promoter strength.
    • This method provides a powerful tool for enriching and identifying functional promoter sequences, applicable in various molecular biology research areas.