**1. Introduction**

Although first models assuming that chromatin in the interphase nucleus is well organized in distinct territories and domains, can be found in the late 19th and early 20th centuries [1, 2], experimental methods of visualization of genome architecture

were missing until the 1970s/1980s [3]. With the breakthrough of three-dimensional (3D) light microscopy, especially 3D fluorescence confocal laser scanning microscopy also developments of specific labeling techniques like fluorescence in situ hybridization (FISH; for review see [4]) started their story of success in genome research and medical diagnostics.

FISH is based on the principle that a DNA probe either amplified in bacteria or by PCR represents the nucleic acid sequence of a given target DNA in the cell nucleus or of a metaphase chromosome [5]. Such a probe has to be thermally or chemically denatured into single DNA strands (if not synthesized by PCR) that can bind complementary to the single, that is, denatured DNA strands of given targets [6, 7]. The probes are labeled with fluorochromes. If these single-stranded probe molecules are added in excess to the denatured target strands, they specifically bind to their complementary target DNA so that a DNA–DNA hybrid with fluorescence labeling is formed [8]. Using various probes labeled with fluorochromes of different colors, multi-target visualization can be processed simultaneously [5].

With automated methods for artificial synthesis of high-purity DNA or PNA [9] oligonucleotides, probe sets of custom-made oligonucleotides have become available for many genomic target sites. Also, highly repetitive sequences in centromeres or telomeres were labeled [10]. By means of so-called "oligopaint" probes (fluorescentlylabeled single-stranded DNA oligonucleotides) covering target sites by huge amounts of oligonucleotides, sequential, highly specific labeling of various targets from 5 kb up to a few Mb was performed. Oligopaint procedures depend on molecular biology techniques while COMBO-FISH probe design is fully based on computer database investigations. Further details of oligopaint can be found in References [11–14].

Standard FISH probes as well as oligopainting probes work on probe-target base-pairing according to the Watson–Crick binding scheme, that is, the singlestranded probe complementarily binds to one target strand. This requires heat or chemical denaturation of the complete DNA in a cell nucleus [6, 7] which could impact the preservation of chromosome morphology and especially chromatin nano-structure [15]. In addition, FISH under vital conditions appears to be nearly impossible.

PNA oligonucleotide probes show a higher target affinity than DNA probes. This allows PNA probes sufficient access to DNA targets without additional heat denaturation, because native chromatin acts in an equilibrium state of single- and doublestrand conformation [16]. Experimentally, this was only shown for repetitive DNA targets [17] but not in oligopainting experiments of complex targets.

In principle, COMBinatorial Oligonucleotide FISH (COMBO-FISH) potentially has several advantages over standard FISH and could also overcome all such drawbacks mentioned above. COMBO-FISH has been invented in the late 1990s [18] and experimentally realized in the early 2000s [19]. It only uses a few short oligonucleotides for specific labeling of a target. These COMBO-FISH probes can be synthesized as DNA or PNA sequences binding complementarily either as a Watson–Crick double-strand or as a Hoogsteen triple-strand [20–22] (**Figure 1**). A low number of probes labeling a target strand reduces synthesis costs and (triple)-strand binding without a strong denaturation step conserves chromatin morphology and organization with the nowadays advantage that the native chromatin structure can be analyzed on the nano-scale by super-resolution localization microscopy in 3D conserved cell nuclei [23–25].

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

#### **Figure 1.**

*Snapshot of a molecular dynamics simulation showing Watson–Crick double-strand pairing and triplex structures of the two classical Hoogsteen pairs for parallel binding: C + \*GC (left) and T\*AT (right). Note: This figure was originally published in [20] and is reproduced with general permission of the publisher.*
