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Improved sandwich-hybridization assay for an electrical DNA-chip-based monitoring of bioprocess-relevant marker genes

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Abstract

Recently, it was shown that electrical DNA-chips in connection with a magnetic bead-based sandwich-hybridization assay can be a suitable alternative for the analysis of gene expression by monitoring the respective mRNA levels. In this study, we established an improved assay which allowed for a significantly shortened but sensitive detection of specific mRNAs. These improvements include the change to a solution-based sandwich-hybridization and the rearrangement of the used oligonucleotide probes. The introduction of a second labeled detection probe further increased the hybridization signals and, in turn, leads to a substantial time reduction of the detection protocol. The presented solution-based sandwich-hybridization protocol for the electrochemical detection of specific mRNAs requires about 60 min and the whole procedure, including sampling, cell disruption, and RNA isolation, approx. 80 min. The assay of this study was verified by nutrient-limited growth experiments and the analysis of selected starvation marker genes of the industrial host Bacillus licheniformis. The expression profiles determined with the electrical chip and the optimized protocol were, in most cases, comparable with the profiles determined by real-time RT-PCR measurements.

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Acknowledgments

This work was supported by the German Federal Ministry of Education and Research (BMBF: 0312707 and 0313751) and by the Ministry of Education, Science and Culture of Mecklenburg-Vorpommern (1400/0052 1401). We thank Stefanie Leja for excellent technical assistance and support during the fermentations.

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Authors and Affiliations

  1. Pharmazeutische Biotechnologie, Institut für Pharmazie, Ernst-Moritz-Arndt Universität, Friedrich-Ludwig-Jahn-Straße 17, 17487, Greifswald, Germany

    Daniel Pioch, Britta Jürgen & Thomas Schweder

  2. Henkel KGaA, VTB Enzymtechnologie, Düsseldorf, Germany

    Stefan Evers & Karl-Heinz Maurer

  3. Institut für Mikrobiologie, Ernst-Moritz-Arndt Universität, Greifswald, Germany

    Michael Hecker

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  1. Daniel Pioch
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  2. Britta Jürgen
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  3. Stefan Evers
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  4. Karl-Heinz Maurer
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  5. Michael Hecker
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  6. Thomas Schweder
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Correspondence to Thomas Schweder.

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Pioch, D., Jürgen, B., Evers, S. et al. Improved sandwich-hybridization assay for an electrical DNA-chip-based monitoring of bioprocess-relevant marker genes. Appl Microbiol Biotechnol 78, 719–728 (2008). https://doi.org/10.1007/s00253-008-1347-z

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  • DOI: https://doi.org/10.1007/s00253-008-1347-z

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