Abstract
Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH) is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as “database mining”. Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols.
Highlights
Errors during mitotic cell division may lead to chromosome mis-segregation
The development of probes that bind to individual human chromosomes depends on the type of DNA repeat sequences identified on the target chromosome
In humans, where imprinting of gonosomal genes exists and dosage-compensation appears to exist for only a small subset of genes, the presence of an extra sex chromosome may lead to clinically recognizable phenotypes, including Turner and Klinefelter syndrome, hypogonadism, etc. [9,46,47]
Summary
Errors during mitotic cell division may lead to chromosome mis-segregation. Aneuploid daughter cells can have severe consequences, for the affected cell, but for an organism as a whole. To facilitate the broad distribution of molecular cytogenetic assays and make DNA probes, as well as multiplex FISH tests, available to the less experienced laboratory, we have undertaken probe production pilot studies, which take advantage of the vast resources generated in the course of the Human Genome Project, such as physical maps and recombinant DNA libraries. Our initial studies focused on the preparation of novel DNA probes for chromosome scoring or “enumeration” in interphase cell nuclei and metaphase spreads, since these seem to remain the most common applications in research and the clinic [30,39]. To the best of our knowledge, the procedures described in the present communication allow a laboratory with common equipment to prepare specific DNA probes in just a few days and, represent the most efficient, rapid and cost-conscious approach to the generation of chromosome-specific DNA probes
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.