DNA Fiber-FISH Staining Mechanism
DNA Fiber-FISH Staining Mechanism
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DNA Fiber-FISH 染色机制
DOI：
10.1177/002215540004800602
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发表时间：
2000
影响因子：
3.2
通讯作者：
A. Raap
中科院分区：
文献类型：
--
作者：
F. M. van de Rijke;R. Florijn;H. Tanke;A. Raap
DNA纤维荧光原位杂交(FISH)是一种高分辨率、范围广泛的物理DNA定位方法，在病理性基因重排的研究中得到了越来越多的应用。在这里，我们介绍的实验旨在了解光纤-FISH后出现的不连续FISH信号模式的性质。一种新的基于顺铂的化学标记方法的使用使我们能够生产完整的生物标记的粘粒靶DNA分子。我们通过免疫荧光监测了这些粘粒靶标在变性和杂交过程中的命运。用缺口平移的地高辛标记的同一粘粒DNA分析不同颜色的FISH探针信号分布。探针信号被证明是变性和杂交后残留的靶信号的子集。我们认为，Fibre-FISH中探针信号的不连续主要是由于靶DNA的丢失和原位复性和附着导致的可及性有限造成的。此外，我们得出结论，FISH的敏感性取决于杂交效率，而不是从小探针产生足够信号的能力。
Fluorescence in situ hybridization to DNA fibers (Fiber-FISH) is a high-resolution, wide-ranging physical DNA mapping method that finds increasing application in the study of pathological gene rearrangements. Here we present experiments designed to understand the nature of the discontinuous FISH signal patterns seen after Fiber-FISH. Use of a novel cisplatin-based chemical labeling method enabled us to produce intact biotinlabeled cosmid target DNA molecules. We monitored by immunofluorescence the fate of such cosmid targets during denaturation and hybridization. The same cosmid DNA labeled with digoxigenin by nick-translation was used to analyze the FISH probe signal distribution in a different color. The probe signals proved to be a subset of the target signals remaining after denaturation and hybridization. We argue that the discontinuity of probe signals in Fiber-FISH is mainly caused by loss of target DNA and limited accessibility due to in situ renaturation and attachment. Furthermore, we conclude that FISH sensitivity is determined by hybridization efficiency and not the ability to generate sufficient signal from small probes.