Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis., The spatial organization of the genome within the nucleus impacts many processes. Here the authors combine oligo-based DNA FISH with single-molecule super-resolution microscopy to image single-copy genomic regions and, taking advantage of SNPs, distinguish allelic regions of homologous chromosomes.

Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

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.

Steps of myTags Custom production using the Indexed Synthesis technique:

1. Probes are designed for each target region separately

2. All probe sequences (up to 27K total) are synthesized together

3. Using unique indexes, each individual probeset is separated into individual oligo pools via PCR deconvolution

4. Composition of oligo pools is verified with next-generation sequencing (NGS)

Note: Short index sequences remain present on the final probe oligos, but are short and not expected to interfere with downstream (F)ISH protocols.

We offer a range of available fluorescent and non-fluorescent label options; please contact us for current available options.

To better suit YOUR experimental needs, we now offer labeling as a service for our wide range of fluorescent and non-fluorescent label options, which can be applied to any of our upstream immortal pool synthesis products. This enables maximum flexibility to meet your dynamic research needs, such as the same probe oligos in multiple colors/tags or a new color/tag of a previously-ordered design.

Our new Labeling Service pairs perfectly with our new Indexed Synthesis options to create a fully custom, ready-to-use toolkit for your complex genome/transcriptome visualization projects.

We may be able to accommodate other labeling options, please contact us for availability.

The number of assays per library depends on a number of factors including the probe density of your library, the size of your target region, the number of probes in the library, and the FISH protocol. Generally we recommend starting with 10pmol of labeled probes per standard FISH slide and then modifying the input amount based on the initial results.

The latest recommended protocols for both labeled and immortal probe libraries can be found in the Resources tab, but in general, myTags FISH libraries are compatible with most FISH protocols. Please contact us for specific recommendations.

We can often accommodate customer-designed probes into the myTags labeling framework. Please contact us for recommendations on the design parameters and other information before designing your probe sequences.

Yes, we can synthesize immortal probe libraries that can be labeled using the Oligopaints labeling method. Please note these probe libraries are not compatible with the standard myTags labeling protocol due to sequence requirements of the Oligopaints method.