Localization of single-copy T-DNA insertion in transgenic shallots (Allium cepa) by using ultra-sensitive FISH with tyramide signal amplification.
The sensitivity of fluorescence in situ hybridization (FISH) for mapping plant chromosomes of single-copy DNA sequences is limited. We have adapted for plant cytogenetics a new signal-amplification method termed tyramide-FISH (Tyr-FISH). Until present this technique has only been applied to human chromosomes. The method is based on enzymatic deposition of fluorochrome-conjugated tyramide. With Tyr-FISH it was possible to detect target T-DNA sequences on plant metaphase chromosomes as small as 710 bp without using a cooled CCD camera. Short detection time and high sensitivity, in combination with a low background, make the Tyr-FISH method very suitable for routine application in plant cytogenetic research. With Tyr-FISH we analysed the position of T-DNA inserts in transgenic shallots. We found that the inserts were preferentially located in the distal region of metaphase chromosomes. Sequential fluorescence in situ hybridization with a 375 bp satellite sequence suggested that a specific T-DNA insert was located within the satellite sequence hybridization region on a metaphase chromosome. Analysis of less-condensed prophase and interphase chromosomes revealed that the T-DNA was integrated outside the satellite DNA-hybridization region in a more proximal euchromatin region.
Khrustaleva
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Plant Research International, Wageningen University and Research Center, PO Box 16, 6700 AA Wageningen, The Netherlands.
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D002874:Chromosome Mapping / Q000379:methods*; D002875:Chromosomes / Q000235:genetics; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D017343:Genes, Plant; D005819:Genetic Markers; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008677:Metaphase; D019697:Onions / Q000235:genetics* / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D019076:Transgenes; D014439:Tyramine / Q000031:analogs & derivatives
D015342:DNA Probes; D004269:DNA, Bacterial; D005819:Genetic Markers; C036483:T-DNA; D014439:Tyramine