Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

Abstract

A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide-and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.T he role of chromosome positioning in gene regulation and chromosome stability is fueling a growing interest in technologies that reveal the in situ organization of the genome. Among these technologies are chromosome conformation capture (3C) (1) and its several iterations, such as Hi-C (2), which are applied to populations of nuclei to identify chromosomal regions that are in close proximity to each other (3, 4). Another technology is fluorescence in situ hybridization (FISH), wherein nucleic acids are targeted by fluorescently labeled probes and then visualized via microscopy; this technology is an extension of methods that once used radioactive probes and autoradiography but have since been adapted to use nonradioactive labels (5-11). FISH is a single-cell assay, making it especially powerful for the detection of rare events that might otherwise be lost in mixed or asynchronous populations of cells. In addition, because FISH is applied to fixed cells, it can reveal the positioning of chromosomes relative to nuclear, cytoplasmic, and even tissue structures. FISH can also be used to visualize RNA, permitting the simultaneous assessment of gene expression, chromosome position, and protein localization.FISH probes are typically derived from cloned genomic regions or flow-sorted chromosomes, which are labeled directly via nick translation or PCR in the presence of fluorophore-conjugated nucleotides or labeled indirectly with nucleotide-conjugated haptens, such as biotin and digoxigenin, and then visualized with secondary detection reagents. Probe DNA is often fragmented into ∼150-to 250-bp pieces to facilitate its penetration into fixed cells (12) and, as many genomic clones contain repetitive sequences that occur abundantly in the genome, hybridization is typically performed in the presence of unlabeled repetitive DNA (13). Another limitation to clone-based probes is that the genomic regions that can be visualized with them are restricted by the availability of clones and the size of ...

Chromosome-Specific Painting in Cucumis Species Using Bulked Oligonucleotides

“…However, a high density of oligos should be considered for probes to detect small chromosomal regions. Beliveau et al (2012) used a probe with .18 oligos per kilobase to detect a 10-kb region on human chromosomes. We also noted that the probe with 7.3 oligos per kilobase produced slightly stronger signals on cucumber pachytene chromosomes than the probe with 3.2 oligos per kilobase.…”

“…The massively synthesized oligos have been successfully labeled as FISH probes in mammalian and Drosophila species (Boyle et al 2011;Yamada et al 2011;Beliveau et al 2012). This approach has opened a new door to develop chromosome-specific painting probes in plants.…”

CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells

“…Despite continuous improvements, DNA FISH requires harsh treatments of heat and formamide to denature dsDNA to allow probe hybridization, thus running the risk of affecting the integrity of the biological structure and genome organization. DNA FISH also is limited in detection resolution with BAC probes and by the high cost of oligo probes (3). Thus there is an opportunity to develop simpler, more efficient and robust in situ imaging of cellular DNA.…”

Spectral imaging to visualize higher-order genomic organization

“…This technique is also amenable to the use of plasmids, Oligopaint-derived probe libraries 44 and high-throughput oligo synthesis strategies. 45 We strongly recommend that researchers confirm the specificity of their probes using PCR, metaphase spreads and localization in diploid cells and prior to usage in this protocol.…”