Abstract 328: Complex oligonucleotide libraries enable high-resolution cytogenetic analysis of human and mouse genomes with fluorescence in situ hybridization (FISH)

Abstract

Abstract Cytogenetic analysis of chromosomes using Fluorescence in situ Hybridization (FISH) is a powerful method for the study of chromosome structure, organization, and stability. However, to date, the utility of FISH has largely depended upon access to cloned template DNA used to generate probes. Clones for specific regions are often unavailable, or the genomic template may contain repetitive and other non-informative sequences which can confound FISH analysis. We have leveraged our ability to chemically synthesize oligonucleotides in massively parallel reactions to produce DNA libraries that can be used to generate FISH probes. The sequences of the oligonucleotides in these libraries are selected in silico using empirically determined criteria so as to target only the most informative elements in the genomic region to be analyzed and avoiding any repetitive elements or regions homologous to other non-targeted loci. Our FISH protocol is similar to ones used for FISH with BAC probes. Using our methods, human chromosomal regions as small as 1.8 kb and as large as chromosome arms were successfully visualized in both metaphase and interphase cells. Using our probe design methods, we readily obtained specific and robust signals for chromosomal regions rich in repeats and/or GC content. We also investigated the utility of these probes for studies of mouse chromosomes by generating probes for over ten loci in the mouse genome. All the probes hybridized well using cytologic and cryosectioned samples. In addition, FFPE samples showed promising preliminary results. We have also applied our FISH methods to develop chromosome-specific enumeration probes for the mouse genome at pericentromeric regions. These enumeration probes should be useful for detecting aneuploidies, polyploidies, and other genomic rearrangements that were previously difficult to study at single-cell resolution, particularly in tissue samples. Successful co-hybridization of chromosome enumeration probes and loci-specific probes in mouse intestinal crypt cells allowed for accurate copy number analyses in tissue samples. We are also continuing to explore other breakthrough applications that are enabled by our oligonucleotide-based FISH methodology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 328.