COMBinatorial Oligonucleotide FISH (COMBO FISH) with Uniquely Binding Repetitive DNA Probes.
During the last decade, genome sequence databases of many species have been more and more completed so that it has become possible to further develop a recently established technique of FISH (Fluorescence In Situ Hybridization) called COMBO FISH (COMBinatorial Oligo FISH) In contrast to standard FISH techniques, COMBO FISH makes use of a bioinformatic search in sequence databases for probe design, so that it can be done for any species so far sequenced. In the original approach, oligonucleotide stretches of typical lengths of 15 30 nucleotides were selected in such a way that they only co localize at the given genome target. Typical probe sets of about 20 40 stretches were used to label about 50 250 kb specifically. The probes of different lengths can be composed of purines and pyrimidines, but were often restricted to homo purine or homo pyrimidine probe sets because of the experimental advantage of using a protocol omitting denaturation of the target strand and triple strand binding of the probes. This allows for a better conservation of the 3D folding and arrangement of the genome. With an improved, rigorous genome sequence database analysis and sequence search according to statistical frequency and 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. These probes can be synthesized commercially as DNA or PNA probes with high purity and labeled by fluorescent dye molecules. Here, new protocols are described for purine pyrimidine probes omitting heat treatment for denaturation of the target so that oligonucleotide labeling can also be combined with immune staining by specific antibodies. If the dyes linked to the oligonucleotide stretches undergo reversible photo bleaching (laser induced slow blinking) the labeled cell nuclei can be further subjected to super resolution localization microscopy for complex chromatin architecture research.
Methods in molecular biology