Combinatorial Oligonucleotide FISH (COMBO-FISH): Computer Designed Probe Sets for Microscopy Research of Chromatin in Cell Nuclei

Abstract

Genome sequence databases of many species have been completed so that it is possible to apply an established technique of FISH (Fluorescence In Situ Hybridization) called COMBO-FISH (COMBinatorial Oligonucleotide FISH). It makes use of bioinformatic sequence database search for probe design. Oligonucleotides of typical lengths of 15–30 nucleotides are selected in such a way that they only co-localize at the given genome target. Typical probe sets of 20–40 stretches label about 50–250 kb specifically. The probes are either solely composed of purines or pyrimidines, respectively, for Hoogsteen-type binding, or of purines and pyrimidines together for Watson-Crick type binding. We present probe sets for tumor cell analysis. With an improved sequence database analysis and sequence search according to uniqueness, a novel family of probes repetitively binding to characteristic genome features like SINEs (Short Interspersed Nuclear Elements, e.g., ALU elements), LINEs (Long Interspersed Nuclear Elements, e.g., L1), or centromeres has been developed. All types of probes can be synthesized commercially as DNA or PNA probes, labelled by dye molecules, and specifically attached to the targets for microscopy research. With appropriate dyes labelled, cell nuclei can be subjected to super-resolution localization microscopy.