**Table 6.**

*List of COMBO-FISH probe targets for the CD44 gene (target sequences are written from left to right in the 5*<sup>0</sup> *- XXXX-3*<sup>0</sup> *direction).*

repetition. Considering the probe length of six trinucleotide units together with one dye molecule at one end, the results of SMLM indicated small chromatin loops for the expansion region rearranging chromatin on the nanoscale so that a deactivation could be explained by geometric reasons in the genome architecture [36].

#### **Figure 3.**

*Example fluorescence microscopy images of COMBO-FISH labeling of centromere targets: (A) centromere 17 labeling in a lymphocyte cell nucleus with the repetitively binding probe (Alexa647-5*<sup>0</sup> *-cttctgtcttctttttata-3*<sup>0</sup> *) and (B) with the repetitively binding probe (Alexa488-5*<sup>0</sup> *-tataaaaagaagacagaag-3*<sup>0</sup> *). In (C) an overview image of centromere 9 labeling in lymphocyte cell nuclei with the repetitively binding probe (Alexa546-5*<sup>0</sup>  *aatcaacccgagtgcaat-3*<sup>0</sup> *) is shown indicating a hybridization efficiency better than 90%.*

#### **Figure 4.**

*(A) ALU-distribution along the genome: The intensity of the bars indicates the frequency within a 500 kb section of the given chromosome. Red: Position of the designed 17mer ALU probe. The sequence associated with the ALU probe appears in the entire genome at different frequency densities. Blue: Corresponding positions of the ALU consensus sequence. The number of emergence of the 17mer probe sequence was compared with the density of the ALU consensus sequence by using the program "Repeatmasker" [50]. Green: Distribution of a selected 17mer from the L1 element. Although this 17mer appears very often in the genome, the frequency density is significantly different from the selected ALU consensus 17mer. (B) Examples of SMLM images of cell nuclei after COMBO-FISH labeling with the 17mer ALU probe. Note: (A) was originally published under CC BY license in [48].*

Although established programs for the design of COMBO-FISH probes and probe sets were available [20, 26], novel so-called alignment-free investigations of k-mers, their frequencies and their positioning along the nucleotide sequence of a chromosome [48, 49] have found oligonucleotide probes that uniquely bind in a given repetition rate to chromatin sequences repetitively occurring as interspersed motives [29]. New generations of specific COMBO-FISH probes were elucidated against SINEs (Short Interspersed Nuclear Elements, e.g., ALU elements [32, 48, 50], **Figure 4**), LINEs (Long Interspersed Nuclear Elements, e.g., L1 [32]), or centromeres [44]. With *Combinatorial Oligonucleotide FISH (COMBO-FISH): Computer Designed Probe Sets… DOI: http://dx.doi.org/10.5772/intechopen.108551*

#### **Figure 5.**

*(A) SMLM overlay image of Alu densities (green), a centromere 9 points cluster (red), and overlaying regions (blue). Note: Only one image plane (no projection) is shown, where one centromere 9 is located. A magnified point coordinate representation of the white box is shown below. (B)–(D) Estimates of chromosome 9 architecture by its centromere and genomic Alu. (B) Plot of the raw point matrix obtained from SMLM data of (A). A circular approximation of a chromosome 9 territory (black circle) modeled from the theoretical distribution of Alu elements (blue dots) around the chromosome 9 centromere (red dots). Lower image: Magnification of the region of interest in the upper image. (C) Idiogram of chromosome 9 showing the positional distribution of Alu probe binding sites (red), Alu consensus sequences (blue), and binding sites of a probe against genomic L1 elements (green). (D) The radial distribution of Alu signal points around a centromere 9 cluster centroid averaged over 38 centromere 9 clusters. Note: These figures were originally published under CC BY license in [32].*

these probes, first evaluations of the spatial organization of chromosome 9 were calculated (**Figure 5**) [32].

Using quantitative SMLM, the in such a way designed ALU COMBO-FISH probe has also been successfully applied in extending the standard methods of biological dosimetry, which aims at reconstructing or estimating from chromosome aberrations the dose from former radiation exposure [48, 50]. In addition, a novel improved preparation protocol circumvents any heat treatment for target denaturation so that mixed purine–pyrimidine probes can be used, that usually undergo Watson–Crick double-strand pairing (**Figure 1**). This so-called low-temperature protocol is the prerequisite to combine oligonucleotide-based COMBO-FISH and immunofluorescence staining by means of specific antibodies [29, 48].
