Liehr Thomas, Padutsch Niklas, Kankel Stefanie
Jena University Hospital, Institute of Human Genetics, Friedrich Schiller University, Jena, Germany.
Front Genet. 2025 May 30;16:1569308. doi: 10.3389/fgene.2025.1569308. eCollection 2025.
In molecular cytogenetics, fluorescence hybridization (FISH) is the main technique used. In both research and diagnostics, FISH depends on well-defined and mapped DNA probes that produce brilliant signals with minimal background, visible in metaphases and/or interphases. Such probes are either ready-to-use and commercially available or provided as unlabeled DNA. The latter can be obtained by flow sorting, microdissection, or by cloning DNA segments into appropriate bacterial vectors. Labeling can be done with either nonfluorescent or fluorescent haptens. According to international guidelines, such FISH probes must have a minimum shelf life, which is only between 2 and 3 years in human genetic diagnostics. The Molecular Cytogenetics Laboratory reporting here has been purchasing, producing, using, and storing FISH probes since the 1990s. For this study, the available stock of approximately 25,000 labeled probes was screened. A total of 581 FISH probes, labeled and approved 1-30 years before reuse, were selected for this study; of these, 75 were commercially available probes labeled 1-20 years ago. All of these probes, stored in the dark at -20°C, worked perfectly well in the FISH method. Although only slight to no differences in exposure times were observed over the years for self-labeled homemade probes, commercial probes labeled with SpectrumOrange had shorter exposure times and maintained them over the years. DNA probes labeled with SpectrumAqua/diethylaminocoumarin showed bright labeling for the first 3 years and then faded. Accordingly, it can be assumed that self-labeled homemade and commercial FISH probes can be stored stably in the dark and at -20°C for at least 30 years or longer. There is no need to test approved probes on a slide after the official expiry date. In practice, this suggests that a FISH probe tube that has been approved can be used in diagnostics until it is empty; there is no need to dispose of these expensive probes at any point due to their age.
在分子细胞遗传学中，荧光原位杂交（FISH）是主要使用的技术。在研究和诊断中，FISH依赖于定义明确且已定位的DNA探针，这些探针能产生清晰的信号且背景极小，在中期和/或间期可见。此类探针要么是即用型且可商购的，要么是以未标记的DNA形式提供。后者可通过流式分选、显微切割或通过将DNA片段克隆到合适的细菌载体中获得。标记可以使用非荧光或荧光半抗原进行。根据国际准则，此类FISH探针必须有最短保质期，在人类遗传诊断中仅为2至3年。此处报告的分子细胞遗传学实验室自20世纪90年代以来一直在购买、生产、使用和储存FISH探针。对于本研究，对约25,000个标记探针的现有库存进行了筛选。总共选择了581个在重新使用前1至30年标记并获批的FISH探针用于本研究；其中75个是1至20年前标记的可商购探针。所有这些探针在-20°C黑暗条件下储存，在FISH方法中效果良好。尽管多年来对于自行标记的自制探针观察到曝光时间仅有轻微差异或无差异，但用SpectrumOrange标记的商业探针曝光时间较短且多年来保持不变。用SpectrumAqua/二乙氨基香豆素标记的DNA探针在最初3年显示出明亮的标记，然后逐渐褪色。因此，可以假设自行标记的自制和商业FISH探针可以在-20°C黑暗条件下稳定储存至少30年或更长时间。在官方过期日期后无需在载玻片上对获批探针进行测试。在实践中，这表明已获批的FISH探针管可用于诊断直至用完；无需因探针的使用年限而在任何时候丢弃这些昂贵的探针。
