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Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

Yang, Kun, Zhang, Hecui, Converse, Richard, Wang, Yong, Rong, Xiaoying, Wu, Zhigang, Luo, Bing, Xue, Liyan, Jian, Li, Zhu, Liquan, Wang, Xiaojia
Plant cell reports 2011 v.30 no.9 pp. 1779-1786
fluorescence in situ hybridization, loci, cell walls, ribosomal DNA, cytoplasm, genes, nucleotide sequences, chromatin, interphase
The compactness of plant chromosomes and the structure of the plant cell wall and cytoplasm provide a great obstacle to fluorescence in situ hybridization (FISH) for single-copy or low-copy DNA sequences. Consequently, many new methods for improving spatial resolution via chromosomal stretching have been employed to overcome this technical challenge. In this article, a technique for extracting cell-wall free nuclei at mitotic interphase, then using these nuclei to prepare extended DNA fibers (EDFs) by the method of a receding interface, whereby slide-mounted chromatin produces EDFs in concert with gravity-assisted buffer flow, was adopted as a result of the low frequency of EDF damage produced by this procedure. To examine the quality of these EDFs, we used single-copy gene encoding S-locus receptor kinase and multi-copy 5S rDNA (ribosomal DNA) as probes. The resulting EDFs proved suitable for high-resolution FISH mapping for repetitive DNA sequences, and the localization of a single-copy locus.