High-resolution <i>in situ</i> hybridization using DNA halo preparations

TLDR

The study aims to improve DNA resolution in fluorescence in situ hybridization. This is achieved by adapting a nuclear extraction technique that produces highly extended DNA loops arranged around the nuclear matrix in a halo‑like structure. The halo technique yields beads‑on‑a‑string hybridization signals, linearizes DNA to ~10 µm, achieves 10–200 kb resolution (potentially a few kb), detects 10 kb overlaps between cosmid clones, requires only 5–10 cells, and is more efficient than pronuclei hybridization, thereby enhancing genome mapping and contig sizing.

Abstract

To improve DNA resolution of fluorescence in situ hybridization we have adapted a nuclear extraction technique, resulting in highly extended DNA loops arranged around the nuclear matrix in a halo-like structure. In situ hybridization signals from alphoid and cosmid DNAs appear as beads-on-a-string, which, according to preliminary experiments, results from the association of individual probe fragments. By multicolor hybridizations we have been able to determine relative map position and to easily detect 10 kb overlap between individual cosmid clones, each of which shows linear beaded signals of ca. 10 μm, suggesting that the DNA is essentially linearized in our protocol. The map configuration can be typically derived from analysis of 5–10 cells only. The resolution range of the technique is at least 10–200 kb, and probably as little as a few kb, thus greatly extending the abilities of the existing FISH methodologies. This novel technique is much more efficient and practicable than pronuclei hybridizations, another method for high resolution FISH, and readily produces results with probes of a variety of genomic origin. In conclusion the DNA halo technique should be able to contribute significantly to the assessment of cosmid and YAC overlaps as well as to the sizing of gaps between adjacent contigs generated in genome projects.