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Fluorescent labelling of in situ hybridisation probes through the copper-catalysed azide-alkyne cycloaddition reaction

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Abstract

In situ hybridisation is a powerful tool to investigate the genome and chromosome architecture. Nick translation (NT) is widely used to label DNA probes for fluorescence in situ hybridisation (FISH). However, NT is limited to the use of long double-stranded DNA and does not allow the labelling of single-stranded and short DNA, e.g. oligonucleotides. An alternative technique is the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC), at which azide and alkyne functional groups react in a multistep process catalysed by copper(I) ions to give 1,4-distributed 1,2,3-triazoles at a high yield (also called ‘click reaction’). We successfully applied this technique to label short single-stranded DNA probes as well as long PCR-derived double-stranded probes and tested them by FISH on plant chromosomes and nuclei. The hybridisation efficiency of differently labelled probes was compared to those obtained by conventional labelling techniques. We show that copper(I)-catalysed azide-alkyne cycloaddition-labelled probes are reliable tools to detect different types of repetitive sequences on chromosomes opening new promising routes for the detection of single copy gene. Moreover, a combination of FISH using such probes with other techniques, e.g. immunohistochemistry (IHC) and cell proliferation assays using 5-ethynyl-deoxyuridine, is herein shown to be easily feasible.

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Abbreviations

FISH:

Fluorescence in situ hybridisation

NT:

Nick translation

dNTP:

Deoxynucleoside triphosphates

CuAAC:

Cu(I)-catalysed azide-alkyne cycloaddition

EdU:

5-Ethynyl-deoxyuridine

PCR:

Polymerase chain reaction

DNA:

Deoxyribonucleic acid

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Acknowledgments

We would like to thank K. Riha for providing different A. thaliana ecotypes. This work was partially supported - VC’s founding - by the European Commission under the Seventh Framework Programme (FP7), as part of the Marie Curie Initial Training Network, EScoDNA (GA no. 317110).

Authors’ contributions

SH conceived and designed the study, performed experiments, analysed data and wrote the manuscript. AM and VC synthesised and provided all of the alkyne-modified probes, conceived the study and assisted with preparing the manuscript. LM and NR performed some experiments. JF conceived the study and assisted with preparing the manuscript. AH conceived the study and wrote the manuscript.

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Authors and Affiliations

  1. Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben, 06466, Stadt Seeland, Germany

    Susann Hesse, Jörg Fuchs, Lu Ma, Nada Raddaoui & Andreas Houben

  2. baseclick GmbH, Floriansbogen 2-4, 82061, Neuried, Germany

    Antonio Manetto, Valentina Cassinelli & Nada Raddaoui

Authors
  1. Susann Hesse
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  2. Antonio Manetto
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  3. Valentina Cassinelli
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  4. Jörg Fuchs
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  5. Lu Ma
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  6. Nada Raddaoui
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  7. Andreas Houben
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Corresponding author

Correspondence to Andreas Houben.

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The authors declare that they have no conflict of interest.

Ethical standards

All performed experiments comply with the current laws of the country in which they were performed. This article does not contain any studies with human or animal subjects performed by any of the authors.

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Responsible Editor: Hans de Jong

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Hesse, S., Manetto, A., Cassinelli, V. et al. Fluorescent labelling of in situ hybridisation probes through the copper-catalysed azide-alkyne cycloaddition reaction. Chromosome Res 24, 299–307 (2016). https://doi.org/10.1007/s10577-016-9522-z

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  • DOI: https://doi.org/10.1007/s10577-016-9522-z

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