Florijn R J, Bonden L A, Vrolijk H, Wiegant J, Vaandrager J W, Baas F, den Dunnen J T, Tanke H J, van Ommen G J, Raap A K
Department of Cytochemistry and Cytometry, State University Leiden, The Netherlands.
Hum Mol Genet. 1995 May;4(5):831-6. doi: 10.1093/hmg/4.5.831.
We have applied two-colour fluorescence in situ hybridization (FISH) to DNA fibers and combined it with digital imaging microscopy for the mapping of large cosmid contigs. The technique was validated using a set of unique plasmids and a cosmid contig both originating from the thyroglobulin (Tg) gene and previously mapped by restriction analysis. The resolution proved to be close to the theoretical lower limit of approximately 1 kb, ranging > or = 400 kb. Subsequently a 400 kb cosmid contig derived from a DMD-YAC was directly mapped by Fiber-FISH. The resulting map is in full agreement with the restriction map. Two-colour Fiber-FISH mapping thus showed to be capable for accurately sizing gaps and overlaps, and to identify chimeric or repeat sequence containing cosmids across a 400 kb region at once. The generated 400 kb 'colour bar-code' was subsequently used to map two DMD deletion breakpoints in patient DNA with an accuracy of 1-2 kb. The results underscore the value of this method for the delineation of chromosomal rearrangements for positional cloning and single patient clinical studies.
我们已将双色荧光原位杂交（FISH）应用于DNA纤维，并将其与数字成像显微镜相结合用于大片段黏粒重叠群的定位。使用一组均源自甲状腺球蛋白（Tg）基因且先前已通过限制性分析定位的独特质粒和一个黏粒重叠群对该技术进行了验证。结果表明，其分辨率接近约1 kb的理论下限，范围大于或等于400 kb。随后，通过纤维FISH直接定位了源自DMD - YAC的一个400 kb黏粒重叠群。所得图谱与限制性图谱完全一致。因此，双色纤维FISH定位能够准确确定间隙和重叠的大小，并能一次性识别跨越400 kb区域的含有嵌合或重复序列的黏粒。随后，所生成的400 kb“彩色条形码”被用于定位患者DNA中的两个DMD缺失断点，精度达到1 - 2 kb。这些结果强调了该方法在定位克隆和单患者临床研究中描绘染色体重排的价值。
