Haar F M, Durm M, Aldinger K, Celeda D, Hausmann M, Ludwig H, Cremer C
University of Heidelberg, FRG.
Biotechniques. 1994 Aug;17(2):346-8, 350-3.
Results of quantitative microscopy for fluorescence in situ hybridization (FISH) signals with repetitive DNA probes (pUC 1.77 and D15Z1) are reported. A nonenzymatic hybridization technique was applied using fluorescein-12-dUTP labeled DNA probes and a buffer system not containing any formamide or equivalent chemical denaturing agents. Following thermal denaturation, the renaturation time was reduced to less than 30 min. The number of wash steps was reduced to one. For the pUC 1.77 probe, the major binding sites (chromosome 1) were distinguished from the minor binding sites by means of fluorescence intensity and spot size. The intensity variation of the two brightest FISH spots (major binding sites) in the same metaphase was 19% for 15 min renaturation time and 16% for 30 min renaturation time. For the D15Z1 probe, generally four bright spots were visible and tentatively assigned according to chromosome length and centromere position (chromosomes 15 and 9). The intensity variation of each two homologues in the same metaphase spread showed a coefficient of variation of 47% (15 min) and 22% (30 min) for chromosome 15, and 19% (15 min) and 15% (30 min) for chromosome 9. The results indicate that the applied technique can considerably accelerate the FISH procedure and is suited for quantitative microscopy.
报告了使用重复DNA探针（pUC 1.77和D15Z1）进行荧光原位杂交（FISH）信号的定量显微镜检查结果。采用了一种非酶杂交技术，使用荧光素-12-dUTP标记的DNA探针和不含任何甲酰胺或等效化学变性剂的缓冲系统。热变性后，复性时间缩短至不到30分钟。洗涤步骤减少到一步。对于pUC 1.77探针，通过荧光强度和斑点大小区分主要结合位点（1号染色体）和次要结合位点。在同一中期，两个最亮的FISH斑点（主要结合位点）的强度变化在复性15分钟时为19%，复性30分钟时为16%。对于D15Z1探针，通常可见四个亮点，并根据染色体长度和着丝粒位置（15号和9号染色体）进行初步定位。在同一中期铺展中，15号染色体每两个同源染色体的强度变化变异系数在复性15分钟时为47%，复性30分钟时为22%；9号染色体在复性15分钟时为19%，复性30分钟时为15%。结果表明，所应用的技术可以显著加速FISH程序，适用于定量显微镜检查。
