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

Author:
Yang, Kun, Zhang, Hecui, Converse, Richard, Wang, Yong, Rong, Xiaoying, Wu, Zhigang, Luo, Bing, Xue, Liyan, Jian, Li, Zhu, Liquan, Wang, Xiaojia
Source:
Plant cell reports 2011 v.30 no.9 pp. 1779-1786
ISSN:
0721-7714
Subject:
fluorescence in situ hybridization, loci, cell walls, ribosomal DNA, cytoplasm, genes, nucleotide sequences, chromatin, interphase
Abstract:
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.
Agid:
451730