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Nicking enzyme–based internal labeling of DNA at multiple loci

Nature Protocols volume 7, pages 643–653 (2012)Cite this article

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Abstract

The labeling of biomolecules has become standard practice in molecular biosciences. Modifications are used for detection, sorting and isolation of small molecules, complexes and entire cells. We have recently reported a method for introducing internal chemical and structural modifications into kbp-sized DNA target substrates that are frequently used in single-molecule experiments. It makes use of nicking enzymes that create single-stranded DNA gaps, which can be subsequently filled with labeled oligonucleotides. Here we provide a detailed protocol and further expand this method. We show that modifications can be introduced at distant loci within one molecule in a simple one-pot reaction. In addition, we achieve labeling on both strands at a specific locus, as demonstrated by Förster resonance energy transfer (FRET) experiments. The protocol requires an initial cloning of the target substrate (3–5 d), whereas the labeling itself takes 4–6 h. More elaborate purification and verification of label incorporation requires 2 h for each method.

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Figure 1: Workflow for internal DNA labeling using a nicking enzyme repeat sequence.
Figure 2: Plasmid map.
Figure 3: Multilocus labeling.
Figure 4: Analysis of second-strand labeling by FRET.

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Acknowledgements

This work was supported by grant SE 1646/2-1 from the Deutsche Forschungsgemeinschaft (DFG) and a starting grant from the European Research Council (no. 261224) to R.S. We gratefully acknowledge L. Pönitz and C. Scholz for technical assistance; H. Brutzer, D. Kauert and D. Klaue for critical reading of the manuscript; and W. Staroske and M. Burkhardt for support in performing single-molecule FRET experiments.

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  1. Biotechnology Center, Dresden University of Technology, Dresden, Germany

    Nicholas Luzzietti, Sabine Knappe, Isabel Richter & Ralf Seidel

Authors
  1. Nicholas Luzzietti
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  2. Sabine Knappe
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  3. Isabel Richter
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  4. Ralf Seidel
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Contributions

N.L., S.K. and I.R. conducted the experiments; N.L. and R.S. designed the experiments and wrote the manuscript.

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Correspondence to Ralf Seidel.

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The authors declare no competing financial interests.

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Luzzietti, N., Knappe, S., Richter, I. et al. Nicking enzyme–based internal labeling of DNA at multiple loci. Nat Protoc 7, 643–653 (2012). https://doi.org/10.1038/nprot.2012.008

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