Haar F M, Durm M, Hausmann M, Ludwig H, Cremer C
Institute of Applied Physics, University of Heidelberg, Germany.
J Biochem Biophys Methods. 1996 Oct 15;33(1):43-54. doi: 10.1016/0165-022x(96)00015-2.
It has been shown for several highly repetitive DNA probes that the newly introduced Fast-FISH (fast-fluorescence in situ hybridization) technique is well suited for quantitative microscopy. The advantage of omitting denaturing chemical agents (e.g., formamide) in the hybridization buffer results in a short hybridization time and a considerable reduction of the number of subsequent washing steps. Choosing the appropriate hybridization temperature and time allows to clearly discriminate major and minor binding sites by quantitative fluorescence microscopy. To further optimize the procedure with reference to reproducibility, a fully programmable thermal-cycler was applied for thermal de- and renaturation. Here, the optimized renaturation conditions for two commercially available alpha-satellite probes (specific for chromosomes 1 and X) are described. For the Boehringer chromosome-1-specific DNA probe, two highly fluorescent binding sites were obtained for 72 degrees C hybridization temperature and 60 min hybridization time. For the Oncor chromosome-X-specific DNA probe, the optimal conditions were found at 74 degrees C and 60 min hybridization time. In both cases the major binding sites were clearly discriminated from only a few weakly fluorescent minor binding sites on metaphase spreads as well as in interphase cell nuclei.
对于几种高度重复的DNA探针而言，新引入的快速荧光原位杂交（Fast-FISH）技术已被证明非常适合定量显微镜观察。杂交缓冲液中省去变性化学试剂（如甲酰胺）的优点是杂交时间短，后续洗涤步骤数量大幅减少。选择合适的杂交温度和时间能够通过定量荧光显微镜清晰区分主要和次要结合位点。为了进一步优化该程序的可重复性，使用了完全可编程的热循环仪进行热变性和复性。在此，描述了两种市售α卫星探针（分别针对1号染色体和X染色体）的优化复性条件。对于勃林格公司的1号染色体特异性DNA探针，在杂交温度72℃和杂交时间60分钟的条件下获得了两个高度荧光结合位点。对于昂科公司的X染色体特异性DNA探针，最佳条件是在74℃和60分钟杂交时间。在这两种情况下，中期染色体铺展以及间期细胞核中的主要结合位点都能与仅有的少数弱荧光次要结合位点清晰区分开来。
