Amal M. Mohamed, Maha Eid, Ola Eid, Shymaa H Hussein, Wael Mahmoud, Rana Mahrous, Khaled Rafaat, Marwa Farid
{"title":"Generation of Dual-Color FISH probes targeting 9p21, Xp21, and 17p13.1 loci as diagnostic markers for some genetic disorders and cancer in Egypt","authors":"Amal M. Mohamed, Maha Eid, Ola Eid, Shymaa H Hussein, Wael Mahmoud, Rana Mahrous, Khaled Rafaat, Marwa Farid","doi":"10.1016/j.jgeb.2024.100449","DOIUrl":null,"url":null,"abstract":"<div><h3>Introduction</h3><div>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.</div></div><div><h3>Aim</h3><div>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.</div></div><div><h3>Methods</h3><div>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.</div></div><div><h3>Results</h3><div>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.</div></div><div><h3>Conclusion</h3><div>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.</div></div>","PeriodicalId":53463,"journal":{"name":"Journal of Genetic Engineering and Biotechnology","volume":"23 1","pages":"Article 100449"},"PeriodicalIF":3.5000,"publicationDate":"2024-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Genetic Engineering and Biotechnology","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1687157X24001525","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
Abstract
Introduction
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.
Aim
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.
Methods
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.
Results
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.
Conclusion
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.
期刊介绍:
Journal of genetic engineering and biotechnology is devoted to rapid publication of full-length research papers that leads to significant contribution in advancing knowledge in genetic engineering and biotechnology and provide novel perspectives in this research area. JGEB includes all major themes related to genetic engineering and recombinant DNA. The area of interest of JGEB includes but not restricted to: •Plant genetics •Animal genetics •Bacterial enzymes •Agricultural Biotechnology, •Biochemistry, •Biophysics, •Bioinformatics, •Environmental Biotechnology, •Industrial Biotechnology, •Microbial biotechnology, •Medical Biotechnology, •Bioenergy, Biosafety, •Biosecurity, •Bioethics, •GMOS, •Genomic, •Proteomic JGEB accepts