A versatile genome-scale PCR-based pipeline for high-definition DNA FISH

Abstract

We developed a cost-effective genome-scale PCR-based method for high-definition DNA FISH (HD-FISH). We visualized gene loci with diffraction-limited resolution, chromosomes as spot clusters and single genes together with transcripts by combining HD-FISH with single-molecule RNA FISH. We provide a database of over 4.3 million primer pairs targeting the human and mouse genomes that is readily usable for rapid and flexible generation of probes.

Figure 1

HD-FISH probe design and synthesis. (a) Construction of a database of unique PCR primer pairs and amplicons in human (Hs) and mouse (Mm) genomes. (b) Cumulative frequency of amplicons along the human and mouse genome. In both cases, 90% of 100 kb tiled windows in which the genome is arbitrarily binned contain ≥ 80 amplicons (grey highlight). (c) Example of amplicon density along human chromosome 17. (d) Probe preparation pipeline from primer selection in a region of interest (blue bar) to labeling of PCR products with the desired fluorophore (red spots).

Figure 2

Specificity and sensitivity of HD-FISH. (a) Human HER2 locus (red) visualized in metaphase spreads (left) and HME cells (right) using a 10 kb ETS probe. Blue: DAPI. (b) Distributions of spot counts for three loci on chromosome 17 including HER2, visualized with 10 vs. 3 kb ETS probes, and with a HER2 commercial probe spanning 460 kb. (c) Distribution of spot sizes for the same HER2 probe as in (a). mRNA: diffraction-limited HER2 mRNA molecules detected by smRNA-FISH.

Figure 3

Versatility of HD-FISH. (a) Chr17 spotting with ten alternatively labeled HD-FISH probes in metaphase spreads (left) and HME cells (mid panel and 3D rendering). (b) Spot quantification in HME cells exemplified in (a). (c) Chr17 spotting with sixteen HD-FISH probes (green and magenta) and paint probes (blue) (left: Z-projections; right: 3D rendering). (d) Left: Chr17 volume estimation using spotting (purple) vs. paint signals at different thresholds (brown gradient). Right: range of median values for curves on the left. Purple line: spotting signal median volume (e–f) Visualization and quantification of HER2 loci (magenta) and transcripts (green) in HME cells. (g) HER2 loci (magenta) and transcripts (green) in breast cancer stroma (above dashes) vs. tumor cells (below dashes). n: number of cells analyzed.