Bringing FISH from Slide to Flow: FISHIS a new technique in Flow Molecular Cytogenetics

Chromosomes and nuclei are mostly studied by FISH (Fluorescent In Situ Hybridization) and microscope observation on slide preparations. They can be analyzed and effectively separated according their DNA content by flow sorting, and this possibility has been used to develop the chromosome approach, the main way to get isolated single chromosomes and nuclei for genomics.  An innovative strategy for precisely labeling individual chromosomes in suspension would improve the resolution power of the chromosome approach allowing a new way of characterizing, and therefore flow-sorting, an all new set of chromosomes. A new method called FISHIS, or Fluorescent In Situ Hybridization in Suspension, has been developed which combines the high-throughput and preparative capabilities of flow cytometry and the specificity of FISH labeling required to sort pure samples of chromosomes and nuclei which are similar to each others by DNA content. The standard FISH protocol relies on the heat denaturation of the probe and target DNAs and their annealing under sufficiently stringent conditions to allow a specific and reproducible hybridization. If treated this way, chromosomes in suspension are inclined to dissolve and/or clump together when exposed to the harsh heat denaturation settings required for FISH. FISHIS is an easy method that removes any washing and heat denaturation standard steps, replaced by an alkaline DNA denaturation. Syntetized fluorescent labeled DNA simple sequence repeats (e.g. SSR) are used as one of the standard probes, further simplifying the process for FISHIS labeling. In less than two hours, FISHIS can effectively label chromosomes in suspension allowing to discriminate, purify and flow sort whole homeologus genomes and a number of chromosomes belonging to several Triticeae species (e.g. T. aestivum, T. turgidum, T. monococcum, T. urartu, D. villosum, and Triticale)