**4. COMBO-FISH with repetitively binding, unique single probes, and applications of super-resolution localization microscopy**

The following chapter will focus on further novel developments of COMBO-FISH using probe sets of only one uniquely binding oligonucleotide [29] that binds repetitively to a given target like a centromere so that the merging fluorescence leads to a microscopic signal. In **Figure 3**, examples for centromere 9 [33] and 17 [34] are shown.

COMBO-FISH probes carrying one fluorochrome molecule at one end of each oligonucleotide are ideal nano-probes for Single Molecule Localization Microscopy [23, 24, 43] (SMLM) in order to analyze chromatin structure and architecture on the nano-scale in subchromosomal regions of cell nuclei [32, 43–47].

As an example, multiple copies of a repetitive probe for a tri-nucleotide expansion region were hybridized and analyzed quantitatively in cells with Fragile-X syndrome (FXS) or Martin-Bell-Syndrome. FXS belongs to the group of the so-called "trinucleotide repeat expansion disorders" consisting of the expansion of a trinucleotide frequency ((CGG)n-expansion) in the 5<sup>0</sup> untranslated region of the Fragile-X Mental Retardation 1 gene (FMR1) on the X-chromosome. The enlargement of the CGG triplet-repeat results in a deactivation of the FMR1 gene and mental retardation of the patient. Multiplets of 6 trinucleotide units ((CGG)6 or (CCG)6 probes) were synthesized showing high specificity to the (CGG)-repeat expansion of the FMR1 gene; thereby a minimum of accessory binding sites were found due to the 6-times


*Combinatorial Oligonucleotide FISH (COMBO-FISH): Computer Designed Probe Sets… DOI: http://dx.doi.org/10.5772/intechopen.108551*
