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首页> 外文期刊>Protoplasma: An International Journal of Cell Biology >A simple non-toxic ethylene carbonate fluorescence in situ hybridization (EC-FISH) for simultaneous detection of repetitive DNA sequences and fluorescent bands in plants
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A simple non-toxic ethylene carbonate fluorescence in situ hybridization (EC-FISH) for simultaneous detection of repetitive DNA sequences and fluorescent bands in plants

机译：用于原位杂交（EC鱼）的简单无毒乙烯碳酸酯，用于同时检测植物中重复DNA序列和荧光带的荧光条件

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The major drawbacks of standard plant fluorescence in situ hybridization (FISH) designed for double-stranded DNA probes include requirement for experimentally determined heat denaturation of chromosomes at high temperatures and at least overnight hybridization. Consequently, processing with chromosomal preparations may easily result in heat-induced deterioration of chromosomal structural details, is time-consuming, and involves the use of toxic formamide and formaldehyde. Here, I have described a simple and appealing non-toxic procedure with ethylene carbonate (EC)a formamide-substituting solvent and double-stranded repetitive DNA probes. Applying EC as a component of the hybridization solution at 46 degrees C not only allowed successful overnight hybridization but also gave a possibility to reduce the hybridization time to 3h, hence converting the technique into a 1-day procedure. Importantly, the EC-FISH tended to preserve well chromosome structural details, e.g., DAPI-positive bands, thus facilitating simultaneous FISH mapping and chromosome banding on the same slide. The procedure requires no formaldehyde and RNA-se treatment of chromosomes, and no heat denaturation of chromosomal DNA. The key condition is to obtain high-quality cytoplasm-free preparations. The method was reproducible in all the plants studied (Allium, Nigella, Tradescantia, Vicia), giving a species-specific signal pattern together with clear DAPI bands on chromosomes. The procedure described here is expected to give a positive stimulus for improving gene-mapping approaches in plants.
机译：标准植物荧光原位杂交（鱼类）的主要缺点是用于双链DNA探针的原位杂交（鱼类）包括在高温下实验确定染色体的热变性的要求，并且至少过夜杂交。因此，具有染色体制剂的加工可以容易地导致染色体结构细节的热诱导的劣化，是耗时的，并且涉及使用有毒甲酰胺和甲醛。这里，我描述了一种具有乙烯碳酸亚乙酯（EC）的简单且吸引人的无毒程序，其甲酰胺替代溶剂和双链重复DNA探针。作为杂交溶液的组分在46摄氏度下施加EC不仅允许成功过夜杂交，而且还可以将杂交时间降低至3H，因此将该技术转化为1天的过程。重要的是，EC鱼倾向于保留染色体结构细节，例如DAPI阳性带，从而促进同一载玻片上的同时鱼类映射和染色体带。该程序不需要甲醛和RNA-SE治疗染色体，并且没有热变性染色体DNA。关键条件是获得高质量的细胞质的制剂。该方法在研究的所有植物（葱属，Nigella，Tradescantia，Vicia）中的所有植物中可重现，使特异性的信号模式与染色体上的透明DAPI带一起。这里描述的程序预计会产生用于改善植物中的基因映射方法的正刺激。

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