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

作者：Yang, Kun; Zhang, Hecui; Converse, Richard; Wang, Yong; Rong, Xiaoying; Wu, Zhigang; Luo, Bing; Xue, Liyan; Jian, Li; Zhu, Liquan^*; Wang, Xiaojia

来源：Plant Cell Reports, 2011, 30(9): 1779-1786.

DOI：10.1007/s00299-011-1086-y

• Extended DNA fibers
• Receding interface
• FISH
• Single-copy DNA
• Multi-copy DNA sequences

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.

• 出版日期2011-9
• 单位西南大学