Čeština
EnglishHigh-resolution FISH on super-stretched flow-sorted plant chromosomes
Valárik, Miroslav; Bartoš, Jan; Kovářová, Pavlína; Kubaláková, Marie; de Jong, H.; Doležel, Jaroslav
PLANT JOURNAL 37: 940-950, 2004
Klíčová slova: Chromosome streching; fluorescence in situ hybridization; chromosome sorting
Abstrakt: A novel high-resolution FISH (fluorescence in situ hybridisation) strategy using super stretching flow-sorted plant chromosomes as targets is described. The technique that allows longitudinal extension of chromosomes of more than 100-times its original metaphase size is especially attractive for FISH on plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosome, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol : acetic acid (3:1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea hybridised with 45S and 5S ribosomal DNAs, the [GAA]n microsatellite, the [TTTAGGG]n telomeric repeat, and a BAC clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity 1 kb. Spatial resolution of neighbouring loci was improved down to 70 kb compared 5 ů 10 Mb after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.
DOI:
Autoři z ÚEB: Jan Bartoš, Jaroslav Doležel, Miroslav Valárik
PLANT JOURNAL 37: 940-950, 2004
Klíčová slova: Chromosome streching; fluorescence in situ hybridization; chromosome sorting
Abstrakt: A novel high-resolution FISH (fluorescence in situ hybridisation) strategy using super stretching flow-sorted plant chromosomes as targets is described. The technique that allows longitudinal extension of chromosomes of more than 100-times its original metaphase size is especially attractive for FISH on plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosome, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol : acetic acid (3:1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea hybridised with 45S and 5S ribosomal DNAs, the [GAA]n microsatellite, the [TTTAGGG]n telomeric repeat, and a BAC clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity 1 kb. Spatial resolution of neighbouring loci was improved down to 70 kb compared 5 ů 10 Mb after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.
DOI: