Visualizing Genomes with Oligopaint FISH Probes

Abstract

Oligopaint probes are fluorescently-labeled, single-stranded DNA oligonucleotides that can be used to visualize genomic regions ranging in size from tens of kilobases to many megabases. This unit details how Oligopaint probes can be synthesized using basic molecular biological techniques as well as provides protocols for FISH, 3D-FISH, and sample preparation.

An Overview of Genome Organization and How We Got There: from FISH to Hi-C

“…However, while the CT position tends to be stable within a given cell (167), it can vary significantly from cell to cell, indicating that not all trans contacts are required and that an exact nuclear position may not be essential (128). This is supported by the observation that tethering to the nuclear periphery mostly alters the expression of genes in cis, with little effect on transcription from other chromosomes, even though trans contacts must be altered in these experiments (17). The positions of CTs might be influenced by their internal chromatin organization.…”

“…These observations suggest that inactive chromatin regions, even those that are gene rich, tend to localize at the nuclear periphery or the nucleolus (see below). In fact, simply relocating a given region to the nuclear lamina is often sufficient to reduce gene expression (17,18), but this is not always the case (19).…”

“…The advantages conferred by 2D-over 3D-FISH are clearer visualization and rapid image analysis. This technique has been predominantly used for determining the nuclear location of genes or translocation from the nuclear periphery to the center and vice versa (17,50) and for determining the location of a DNA segment in relation to the rest of the chromosome (45,48,51). We have used 2D-FISH to determine changes in chromatin condensation at the submegabase level, during differentiation and across a polarizing axis during development (45,49) or between wild-type and mutant cells (46,52).…”

In Situ Super-Resolution Imaging of Genomic DNA with OligoSTORM and OligoDNA-PAINT

“…Here we describe how two forms of single-molecule-based super-resolution imaging can be used for the in situ visualization of chromosomal DNA [5, 6]. These technologies, called OligoSTORM and OligoDNA-PAINT, use Oligopaint technology [7, 8] to generate single-stranded oligonucleotide probes for fluorescent in situ hybridization (FISH) and then use the probes in combination with, respectively, Stochastic Optical Reconstruction Microscopy (STORM) [9-11] and DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) [12– 14] to generate high-resolution images of genomic DNA. Below, we provide brief descriptions of Oligopaints, OligoSTORM, and OligoDNA-PAINT, followed by protocols for generating Oligopaint probes from a library of oligonucleotides (oligos) and preparing samples for imaging by OligoSTORM or OligoDNA-PAINT.…”

“…Oligopaints are short single-stranded oligos which, when labeled directly or indirectly with a fluorophore, can be used to visualize genomic regions as small as a few kilobases (kbs) to as large as tens of megabses (Mbs) or more [7, 8]. The oligos feature a short region of genomic homology (typically, 30 to 42 bases) flanked by nongenomic sequences, called Mainstreet and Backstreet, and are generated through amplification of oligo libraries that have been computationally designed and then synthesized by traditional column-based or massively parallel array-based methods – in either case, libraries can be readily purchased from commercial vendors (Fig.…”