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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Optical mapping of site-directed cleavages on single DNA molecules by the RecA-assisted restriction endonuclease technique.
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Optical mapping of site-directed cleavages on single DNA molecules by the RecA-assisted restriction endonuclease technique.

机译：通过RecA辅助的限制性内切核酸酶技术对单个DNA分子上的定点切割进行光学定位。

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摘要

Fluorescence in situ hybridization (FISH) resolution has advanced because newer techniques use increasingly decondensed chromatin. FISH cannot analyze restriction enzyme cutting sites due to limitations of the hybridization and detection technologies. The RecA-assisted restriction endonuclease (RARE) technique cleaves chromosomal DNA at a single EcoRI site within a given gene or selected sequence. We recently described a mapping technique, optical mapping, which uses fluorescence microscopy to produce high-resolution restriction maps rapidly by directly imaging restriction digestion cleavage events occurring on single deproteinized DNA molecules. Ordered maps are then constructed by noting fragment order and size, using several optically based techniques. Since we also wanted to map arbitrary sequences and gene locations, we combined RARE with optical mapping to produce site-specific visible EcoRI restriction cleavage sites on single DNA molecules. Here we describe this combined method, named optical RARE, and its initial application to mapping gene locations on yeast chromosomes.
机译：荧光原位杂交（FISH）分辨率已经提高，因为新技术使用了越来越不浓缩的染色质。由于杂交和检测技术的限制，FISH无法分析限制性酶切位点。 RecA辅助的限制性核酸内切酶（RARE）技术可在给定基因或选定序列内的单个EcoRI位点切割染色体DNA。我们最近描述了一种作图技术，光学作图，它使用荧光显微镜通过直接成像单个脱蛋白的DNA分子上发生的限制性消化切割事件而迅速产生高分辨率的限制性图谱。然后，使用几种基于光学的技术，通过注意片段的顺序和大小来构建有序图。由于我们还想定位任意序列和基因位置，因此我们将RARE与光学定位相结合，以在单个DNA分子上产生特定于位点的可见EcoRI限制性切割位点。在这里，我们描述了这种称为光学RARE的组合方法，并将其最初用于在酵母染色体上定位基因位置。

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