Agilent SureFISH - probes for rapid identification of a wide range of chromosomal aberrations across the genome

Fluorescence in situ Hybridization (FiSH) is a powerful technique for studying the structure, organization, and localization of nucleic acids within individual cells. However, the use of FiSH has often been dependent upon access to cloned template DNA for the generation of probes. Clones for specific regions may be unavailable, or the genomic region of interest may contain repetitive and other non-informative sequences which can be problematic for FiSH analysis. Agilent has leveraged its ability to chemically synthesize DNA in massively parallel reactions to produce libraries of oligonucleotides up to 200 bases in length 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 avoid repetitive elements or regions homologous to other non-targeted loci. Using oligonucleotide library-derived FiSH probes on DNA, human genomic regions as small as 1.8 kb and as large as whole chromosomes can be visualized in both metaphase and interphase cells using the same simple assay protocol. Because of the inherent flexibility in our probe design methods, we readily visualized regions rich in repeats and/or GC content. Using oligonucleotide library-derived FiSH probes on RNA, Agilent has been able to detect the localization of a variety of both coding and non-coding RNAs in fixed cells, using both conventional fluorescence and structured illumi- nation microscopy. Simultaneous hybridization of FiSH probes labeled with different fluorophores enables visualization of multiple sequences at once. Using probes designed specifically to transcribed vs. non-transcribed regions, Agilent has been able to simultaneously detect DNA and RNA from the same locus in the same FiSH assay. Agilent's oligo FiSH methods are readily compatible with the co-detection of cellular proteins by immunocytochemistry. The ability to generate high performance FiSH probes using chemically synthesized oligo libraries that can work flexibly with co-detection of other molecules yields a valuable tool for studies of how localization of specific nucleic acids impacts biological function.