van de Rijke F M, Florijn R J, Tanke H J, Raap A K
Departments of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
J Histochem Cytochem. 2000 Jun;48(6):743-5. doi: 10.1177/002215540004800602.
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 biotin-labeled 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. (J Histochem Cytochem 48:743-745, 2000)
DNA纤维荧光原位杂交技术（Fiber-FISH）是一种高分辨率、广泛应用的物理DNA图谱绘制方法，在病理性基因重排研究中的应用越来越广泛。在此，我们展示了旨在理解Fiber-FISH后出现的不连续FISH信号模式本质的实验。使用一种基于顺铂的新型化学标记方法，使我们能够产生完整的生物素标记的黏粒靶DNA分子。我们通过免疫荧光监测了此类黏粒靶在变性和杂交过程中的命运。用缺口平移法标记地高辛的相同黏粒DNA用于以不同颜色分析FISH探针信号分布。探针信号被证明是变性和杂交后剩余靶信号的一个子集。我们认为，Fiber-FISH中探针信号的不连续性主要是由于靶DNA的丢失以及原位复性和附着导致的可及性受限。此外，我们得出结论，FISH敏感性由杂交效率决定，而非小探针产生足够信号的能力。（《组织化学与细胞化学杂志》48:743 - 745, 2000）
