Heiskanen M, Kallioniemi O, Palotie A
Laboratory Department of Helsinki, University Central Hospital, Finland.
Genet Anal. 1996 Mar;12(5-6):179-84.
One of the most time-consuming steps in positional cloning is the physical mapping of probes from the critical chromosomal region and the assembly of a genomic contig of large insert probes. New high-resolution Fiber-FISH techniques have significantly facilitated this tedious task by enabling rapid direct visualization of the order, degree of overlap and gap sizes of adjacent large insert clones. We have developed a method, where agarose-embedded DNA (PFGE block) is used as a source for preparing linearized DNA targets on microscope slides. This modification of the fiber-FISH technique has been successfully used in physical mapping in the 1-300 kb range as well as for detecting genomic rearrangements. Here, we present a refined protocol of our original technique. The application of this technique to agarose embedded yeast cells is also demonstrated. Finally, critical steps and trouble shooting of the method are addressed.
定位克隆中最耗时的步骤之一是对关键染色体区域的探针进行物理图谱绘制以及构建大插入片段探针的基因组重叠群。新的高分辨率纤维荧光原位杂交（Fiber-FISH）技术通过能够快速直接观察相邻大插入片段克隆的顺序、重叠程度和间隙大小，极大地促进了这项繁琐的任务。我们开发了一种方法，其中将琼脂糖包埋的DNA（脉冲场凝胶电泳（PFGE）块）用作在显微镜载玻片上制备线性化DNA靶标的来源。这种纤维荧光原位杂交技术的改进已成功用于1-300 kb范围内的物理图谱绘制以及检测基因组重排。在此，我们展示了我们原始技术的优化方案。还展示了该技术在琼脂糖包埋酵母细胞中的应用。最后，讨论了该方法的关键步骤和故障排除。
