Souza M M, Urdampilleta J D, Forni-Martins E R
Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Ilhéus, BA, Brasil.
Genet Mol Res. 2010 Nov 3;9(4):2148-55. doi: 10.4238/vol9-4gmr951.
Cytological preparations for the fluorescent in situ hybridization (FISH) technique require cytoplasm-free metaphases, with well-spread chromosomes, for the localization of DNA sequences and chromosome mapping. We tested various procedures for FISH analysis of Passiflora cacaoensis, P. gardneri and hybrid F₁ progeny of P. gardneri x P. gibertii. Two treatments with four enzymes and three incubation times were compared. The material was treated with 1.0 M HCl before enzymatic digestion. The following criteria were used to determine the quality of the metaphases: a) lack or presence of cytoplasm; b) well-spread chromosomes or with overlap; c) complete or incomplete chromosome number (2n). The enzyme Pectinex(®) SP ULTRA gave the best performance, with the shortest incubation time. The best results were observed after 30 min of incubation; more than 70% of the metaphases did not have large amounts of cytoplasm or overlapping chromosomes, and about 75% maintained the chromosome number. FISH was carried out using a 45S rDNA probe (pTa71) labeled with biotin and detected with fluorescein isothiocyanate. Sites with strong staining and without nonspecific signals were observed. Our methodological adaptations allowed the preparation of metaphase slides of high quality for the FISH technique, with less time required for the preparation of samples.
用于荧光原位杂交（FISH）技术的细胞学标本需要无细胞质的中期分裂相，其染色体要充分伸展，以便进行DNA序列定位和染色体图谱绘制。我们测试了多种用于西番莲可可叶、加氏西番莲以及加氏西番莲与吉氏西番莲杂交F₁代的FISH分析程序。比较了用四种酶进行的两种处理以及三种孵育时间。在酶解之前，材料先用1.0 M盐酸处理。采用以下标准来确定中期分裂相的质量：a）有无细胞质；b）染色体充分伸展还是相互重叠；c）染色体数目完整还是不完整（2n）。果胶酶Pectinex(®) SP ULTRA表现最佳，孵育时间最短。孵育30分钟后观察到最佳结果；超过70%的中期分裂相没有大量细胞质或染色体相互重叠的情况，约75%保持了染色体数目。使用用生物素标记并用异硫氰酸荧光素检测的45S rDNA探针（pTa71）进行FISH。观察到有强染色且无非特异性信号的位点。我们的方法改进使得能够制备用于FISH技术的高质量中期分裂相玻片，且样品制备所需时间更短。
