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

The detection of 10 fluorochromes (dye molecules at both ends of each oligonucleotide probe) would require not only a sensitive microscope but also a background free preparation. Since in tumors P53 is inactivated (sometimes associated by a copy number loss of the gene, see, e.g., [40]), the protein MDM2 which can inactivate P53 when overexpressed can be investigated. In cells with an overexpression of MDM2 an extreme inactivation of the tumor suppressor protein can occur via binding of MDM2 to the transactivation domain of TP53. However, the treated gene, MDM2, does not contain sufficient homo-purine/homo-pyrimidine sequences so that a probe set has to be designed with an overlap on neighboring regions (**Table 5**).


#### **Table 5.**

*List of COMBO-FISH probe targets for MDM2 and its surroundings (target sequences are written from left to right in the 5*<sup>0</sup> *-XXXX-3*<sup>0</sup> *direction).*

CD44 is a receptor for hyaluronic acid, which plays an important role in cell migration, tumor growth and progression. Accumulating evidences have shown that the CD44 gene is abundantly expressed in cancer-initiating cells (CICs), and has thus been implicated as a CIC marker [41, 42] in several malignancies of hematopoietic and epithelial origin, including gastric cancer. Moreover, CD44 gene amplification was also found in gastric cancer. **Table 6** shows the targets for an appropriate CD44 gene probe set.

Fusion proteins originate from reciprocal translocations. Sets of oligonucleotides were designed in such a way that translocations get cognizable. There are two breakpoint regions—one on every chromosome. Therefore, four sets of oligonucleotides are needed: One before and after the breakpoint on the two chromosomes. For microscopy labeling with different colors is necessary; the sets of the first chromosome need to be labeled for instance with a red dye and the ones on the second chromosome, for instance, with a green dye. After hybridizing there are the following possible results: (a) Two red spots and two green spots are close together for each color, but the red ones are clearly separated from the green ones. This is the normal case without translocation. (b) There are two parts where one red and one green spot are next to each other. In this case the translocation has occurred: Both chromosomes broke at the major breakpoint and the wrong ends were joined. With four colors more details are visible. For example, when one color is visible on two locations, another breakpoint has been observed.

The minimum requirement for detecting clusters of homo-purine/homopyrimidine sequences on DNA is 6 oligonucleotides within a range of 250 kb. It is not necessary that the oligonucleotides are all located on the breakpoint regions itself. To get bright and emphasized signals, sets with 30 oligonucleotids each were designed for the following examples: ABL - BCR t(9,22)(q34,q11); AML1 - ETO t(8;21)(q22;q22); MYC - IGH t(8,14)(q24,q32); PML - RARA t(15,17)(q22,q21);PLZF - RARA t(11,17) (q23,q21). Since these lists would extend the article to an inacceptable size, the lists will be available from the authors on request.
