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One-step assay for the quantification of T4 DNA ligase

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Abstract

As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6–5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays.

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Acknowledgments

This work was supported by the Federal Ministry of Education and Research (BMBF) GoBio project no. 031598870 to TZ. The authors thank Prof. Ralf Hoffmann for lab access and helpful discussions on the manuscript.

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Author notes

  1. Thole Zuchner

    Present address: Octapharma Biopharmaceuticals GmbH, Im Neuenheimer Feld 590, 69120, Heidelberg, Germany

Authors and Affiliations

  1. Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, (BBZ), Institute of Bioanalytical Chemistry, Leipzig University, Deutscher Platz 5, 04103, Leipzig, Germany

    Steffi Franke, Thomas Kreisig, Karin Buettner & Thole Zuchner

Authors
  1. Steffi Franke
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  2. Thomas Kreisig
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  3. Karin Buettner
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  4. Thole Zuchner
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Correspondence to Thole Zuchner.

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Franke, S., Kreisig, T., Buettner, K. et al. One-step assay for the quantification of T4 DNA ligase. Anal Bioanal Chem 407, 1267–1271 (2015). https://doi.org/10.1007/s00216-014-8351-1

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  • DOI: https://doi.org/10.1007/s00216-014-8351-1

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