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

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• 作者：Susann Hesse ; Antonio Manetto ; Valentina Cassinelli ; Jörg Fuchs…
• 刊名：Chromosome Research
• 出版年：2016
• 出版时间：September 2016
• 年：2016
• 卷：24
• 期：3
• 页码：299-307
• 全文大小：4,462 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Cell Biology
  Human Genetics
  Animal Genetics and Genomics
  Plant Genetics and Genomics
  
• 出版者：Springer Netherlands
• ISSN：1573-6849
• 卷排序：24

文摘

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.KeywordsFISHChromosomesIn situ labellingInterphaseDNA sequence detection