Fluorescent labelling of in situ hybridisation probes through the copper-catalysed azide-alkyne cycloaddition reaction.
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.
Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben, 06466, Stadt Seeland, Germany.
Susann
S
Hesse
baseclick GmbH, Floriansbogen 2-4, 82061, Neuried, Germany.
Antonio
A
Manetto
baseclick GmbH, Floriansbogen 2-4, 82061, Neuried, Germany.
Valentina
V
Cassinelli
Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben, 06466, Stadt Seeland, Germany.
Jörg
J
Fuchs
Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben, 06466, Stadt Seeland, Germany.
Lu
L
Ma
Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben, 06466, Stadt Seeland, Germany.|baseclick GmbH, Floriansbogen 2-4, 82061, Neuried, Germany.
Nada
N
Raddaoui
Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben, 06466, Stadt Seeland, Germany. houben@ipk-gatersleben.de.
Andreas
A
Houben
D000480:Alkynes / Q000737:chemistry*; D017360:Arabidopsis / Q000235:genetics; D001386:Azides / Q000737:chemistry*; D049109:Cell Proliferation / Q000235:genetics; D032461:Chromosomes, Plant / Q000235:genetics*; D003300:Copper / Q000737:chemistry*; D061565:Cycloaddition Reaction / Q000379:methods*; D015342:DNA Probes / Q000737:chemistry*; D003856:Deoxyuracil Nucleotides; D001467:Hordeum / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D012434:Secale / Q000235:genetics; D014908:Triticum / Q000235:genetics
D000480:Alkynes; D001386:Azides; D015342:DNA Probes; D003856:Deoxyuracil Nucleotides; D003300:Copper; C033258:5-ethynyl-2'-deoxyuridylic acid
Chromosomes; DNA sequence detection; FISH; In situ labelling; Interphase