**2. COMBO-FISH: principle and applications**

In contrast to standard FISH where probes are usually cut and amplified by molecular biology techniques, COMBinatorial Oligonucleotide FISH (COMBO-FISH) follows a completely different strategy for probe design [19, 20, 26, 27]. If a genome of a species is sequenced and the sequence is cataloged in a database, oligonucleotide probes can be searched and a set of probes can be designed in silico. Also, their specificity is controlled by computer analysis searching for all possible binding sites of each probe [20, 28, 29].

Any given genome target that should be specifically labeled, can be selected in a DNA sequence database and the numbers of the beginning and end nucleotides are determined exactly. This cannot only be done for human but also for any other species with completely known DNA sequences that can be read in established DNA database archives like NCBI.

The process works as follows [20]: firstly, the beginning and end numbers of a given target (for instance, a gene) are selected. Then oligonucleotide stretches of 15–30 distinct bases are determined in such a way that just the combination of those stretches is singularly co-localizing at the given target site. All binding sites of each probe are determined. Finally, several probe combinations may be excluded from consideration: (a) these are those that have accessory binding sites at other locations in a genome than at the given target site or (b) those that co-localize at several loci in the same genome (**Table 1**). In addition to these principles governing the basic probe selection, further characteristic features (experimental and theoretical) can be taken into account to ensure a stable homogenous hybridization protocol. Among these, the most important ones are oligonucleotide length, binding energy [28], homo-purine/ homo-pyrimidine sequences [30, 31], melting temperature [32], CG-content [32], etc. (see **Table 2** as an example). Typically, such oligonucleotide stretches included in a probe set have a length of 15–30 nucleotides each.

Several probe sets created according to this procedure (e.g., ABL, BCR, Her2neu, GRB7, AMACR, etc., see below) have been published or will be shown in the next chapter.


#### **Table 1.**

*Examples of COMBO-FISH target sites ("Gene"), their positions according to the banding annotation ("Positions on chromosome"), start and end position in the human data base ("Base position"), possible number of probes ("B") and the finally remaining specifically co-localizing probes ("R").*

Finally, the computer-optimized probe set can be synthesized as DNA probes, PNA probes, SMART probes, or TINA (Twisted Intercalating Nuclear Acid) probes with high purity [30, 33–35]. PNA probes have a peptide backbone instead of a sugar– phosphate backbone of DNA. SMART probes also called molecular beacons consist of a stem-loop conformation quenching fluorescence by the closed loop until loop

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


*Note: This table was originally published under CC BY license in [32]. a Melting temperature (median value of the denaturation curve).*

#### **Table 2.**

*Example of a COMBO-FISH probe set for AMACR and some physical values considered in the selection of oligonucleotide stretches.*

opening when probe and target are binding. TINA probes are oligonucleotides with additional anchoring molecules incorporated. Custom made oligonucleotide probes usually carry one dye molecule at one end or both ends each.

Depending on the base composition of the oligonucleotide probes, they can be designed in their 30 –5<sup>0</sup> direction either for Watson–Crick binding (duplex forming probes result in complimentary probe-target double strands) or for Hoogsteen binding (triplex forming probes result in triple strands to homo-purine or homopyrimidine sequences as targets of the intact double strand) (**Figure 1**). COMBO-FISH probes targeting in Watson–Crick configuration are more flexible since they can be designed in a mixture of purine and pyrimidine bases. Hoogsteen binding probes use solely either purines or pyrimidines. It should mentioned that also exceptional (non-homo) Hoogsteen triples exist which can be incorporated into the probe design.

Due to the optical diffraction of a microscope lens, the point image of a fluorescence dye molecule spreads to an image of typically about 250 nm using a high numerical aperture lens. So the fluorescence of a probe combination within a target size less than typically about 250 kb merges into a homogeneous COMBO-FISH "spot". Typical examples are shown in **Figure 2**.

Detailed protocols for COMBO-FISH labeling of blood cells, fibroblasts, tumor culture cells, or tissue cells are described elsewhere [27, 29]. These protocols can be applied for duplex or triplex forming probe sets. COMBO-FISH for labeling of specific gene targets in cell nuclei has been described for several applications as for instance: The gene of the receptor tyrosine kinase 2 (HER2/NEU) [28, 34] (**Figure 2A** and **B**), the gene of the growth factor receptor-bound protein 7 (GRB7) [34], the breakpoint cluster region (BCR) on chromosome 22 [30], the ABL proto-oncogene 1 (ABL) on chromosome 9 [19, 30, 31], and T-box 1(TBX1) [33] (**Figure 2C** and **D**), the promotor region of the FMR1 gene [36] (**Figure 2E**) and the Alpha-Methylacyl-CoA Racemase coding gene (AMACR) on chromosome 5 [32] (**Table 2**; **Figure 2F**). Using the probe set for the ABL gene region and Spatially Modulated Illumination Microscopy [37], significant volume changes of the labeled regions were observed in cell nuclei of CML patients before and after medical treatment [35]. Beyond gene target labeling by probe sets of several

#### **Figure 2.**

*Example fluorescence microscopy images of COMBO-FISH labeling (arrows) of gene targets: (A) HER2/NEU gene labeling in a cell nucleus and (B) on metaphase (combination of 18 oligonucleotide probes); (C) TBX1 gene labeling in a cell nucleus and (D) in an early stage of mitosis (combination of 15 oligonucleotide probes); (E) FMR1 promotor region labeling on chromosome X of a male cell nucleus (combination of 20 oligonucleotide probes); AMACR gene labeling in a cell nucleus (combination of 29 oligonucleotide probes; see Table 2). Note: With the exception of the nucleus in (E), no counterstain was applied.*

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

different probe-sequences co-localizing at a given target only, also unique probes were found that were specifically labeling either interspersed genome regions by one copy each or centromeres by multi-copies (see chapter 4). Such an 18mer oligonucleotide PNA probe repetitively binding on centromere 9 was micro-injected into lymphocyte cell nuclei under in vivo conditions. After further incubation of the labeled cells, the sample was fixed and subjected to microscopy. The results indicated that the probe material was binding to the target region in the nuclei before fixation [33].
