Mohamed Amal M, Eid Maha, Eid Ola, Hussein Shymaa H, Mahmoud Wael, Mahrous Rana, Refaat Khaled, Farid Marwa
Human Genetics Department, Human Genetics and Genome Research Institute, National Research Centre, Egypt.
Human Genetics Department, Human Genetics and Genome Research Institute, National Research Centre, Egypt.
J Genet Eng Biotechnol. 2025 Mar;23(1):100449. doi: 10.1016/j.jgeb.2024.100449. Epub 2024 Dec 18.
The fluorescence in situ hybridization (FISH) is a very important technique, as it can diagnose many genetic disorders and cancers. Molecular cytogenetic analysis (FISH) can diagnose numerical chromosome aberrations, sex chromosomes anomalies, and many genetic disorders.
With the limited number of commercially available probes that do not cover all research needs and the high prices of the commercial probes, our goal is to apply recent technologies to produce FISH probes that can accurately and sensitively diagnose genetic diseases and cancer in Egypt and establishing the inhouse production of different FISH probes. We intend to adhere to the published guidelines and validation procedures to ensure the production of accurate FISH probes for clinical diagnosis.
We used specific DNA segments extracted from BAC clones, and we performed nick translation to label the segment with fluorescence labeled dye. The second method involved the use of specific primers for the centromere of certain chromosomes and using PCR technique for amplification and labeling. The probes were tested on metaphase and interphase cells derived from cultured human peripheral blood samples. We followed standard guidelines to test the adequacy of probe slide hybridization, proper probe localization, probe sensitivity and specificity, probe reproducibility, cut-off values, and overall probe validation.
In this research, we presented the generation of three dual-color probes, each probe has a control locus. We offered three dual-color probes targeted 9p21, Xp21 and 17p13.1 loci. chromosome 9p21probe for diagnosis of structural abnormalities in chromosome 9, the Xp21 to test for structural abnormalities of chromosome X, and the 17p13.1 for TP53 gene to detect the loss of p53. We also produced probes for Down syndrome specific region, Rb gene and centromeres for chromosomes X, 17, and 18.
The produced probes are specific and sensitive and can be produced at the commercial level in the laboratory. The production of FISH probes in Egypt can be used as a powerful diagnostic marker for genetic disorders and cancers and our work can be consider as a base to start national project to produce our needs of FISH probes.
荧光原位杂交（FISH）是一项非常重要的技术，因为它可用于诊断多种遗传疾病和癌症。分子细胞遗传学分析（FISH）能够诊断染色体数目畸变、性染色体异常以及多种遗传疾病。
鉴于市面上可用的探针数量有限，无法满足所有研究需求，且商业探针价格高昂，我们的目标是应用最新技术生产能够准确、灵敏地诊断埃及遗传疾病和癌症的FISH探针，并建立不同FISH探针的内部生产体系。我们打算遵循已发表的指南和验证程序，以确保生产出用于临床诊断的准确FISH探针。
我们使用从BAC克隆中提取的特定DNA片段，并进行缺口平移，用荧光标记染料标记该片段。第二种方法是使用针对某些染色体着丝粒的特异性引物，并利用PCR技术进行扩增和标记。将这些探针在源自培养的人类外周血样本的中期和间期细胞上进行测试。我们遵循标准指南来测试探针玻片杂交的充分性、探针的正确定位、探针的敏感性和特异性、探针的可重复性、临界值以及整体探针验证。
在本研究中，我们展示了三种双色探针 的生成，每种探针都有一个对照位点。我们提供了针对9p21、Xp21和17p13.1位点的三种双色探针。9号染色体9p21探针用于诊断9号染色体结构异常，Xp21用于检测X染色体结构异常，17p13.1用于检测TP53基因以检测p53缺失。我们还生产了唐氏综合征特定区域、Rb基因以及X、17和18号染色体着丝粒的探针。
所生产的探针具有特异性和敏感性，并且可以在实验室实现商业化生产。在埃及生产FISH探针可作为遗传疾病和癌症的有力诊断标志物，我们的工作可被视为启动国家项目以生产我们所需FISH探针的基础。
