Basic steps of fluorescent in situ hybridization technique. If the probes are labeled directly with fluorescent molecules the step (3) is not necessary. 

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... to the advances on molecular techniques to study the eukaryotic genomes, the cytogenetic provided the first information on the genome organization and location of distinct DNA fractions on chromosomes. For several decades these information were obtained using the classical cytogenetic methods that had permitted only the description of general chromosomal characteristics. Besides the use of classical techniques, some studies were conducted through the use of differential staining, such as C-banding, silver nitrate and fluorochrome staining that led to a better characterization of the chromosomes and their content. Despite these advances, the genome structure under the focus of chromosome analysis remained limited. In recent decades, the application of in situ hybridization using DNA probes onto chromosomes caused a cytogenetic revolution, leading to the transition between the “classical cytogenetic era” to the “molecular cytogenetic era”. This methodology has permitted the precise location of specific DNA sequences generating more detailed information concerning chromosomal and genomic organization in several animal groups. Using the in situ assay it is possible to integrate the molecular information of DNA sequences to their physical location along chromosomes of all eukaryotic representatives [1, 2]. The hybridization principle is based in the denaturation of chromosomal DNA and its renaturation under the presence of complementary DNA labeled probes, which have the capacity to anneal in the regions with base pair complementarity [3, 4]. Briefly, the in situ hybridization consists of four steps, (1) denaturation, (2) hybridization, (3) probe detection, and (4) microscopic analysis ( Fig. 1). Since the first application of in situ hybridization in chromosomes, this technique has been suffered modifications related to all steps, involving probe achievement, sensitivity increasing in the probe detection, resolution, specificity and quality of the results. Formerly, the DNA probes for in situ hybridization were labeled using radioactive isotopes as 32 P, 125 I, 3 H and 35 S. On the other hand, nowadays most probes for in situ hybridization are visualized/detected through the use of fluorescent molecules under an epifluorescence microscope and the technique is named as fluorescent in situ hybridization (FISH). In animal chromosomes, the FISH has been applied in the detection of distinct sequences allowing the integration of cytogenetic to linkage maps, analysis of chromosomal structure, genome organization, chromosomal ongoing and evolution. For fish and insect chromosomes, the most applied class of probes is represented by the repeated sequences including multigene families of ribosomal RNA (rRNA) genes and satellite DNAs (satDNA), and , in a lesser extent , transposable elements and microsatellites [for example 5-11]. The repetitive sequences have been obtained by distinct approaches, including for example, polymerase chain reaction (PCR), enzymatic restriction, and chromosome microdissection. Some of the information concerning the type of sequences, methods for probe obtainment, and application of FISH in fish and insect chromosomes will be presented along this chapter, showing the potential of this technique to advance in the knowledge of genome organization and chromosomal differentiation during the evolutionary history of species and groups. The presence of large amount of repetitive DNAs in eukaryotic genomes is a ubiquitous feature and these sequences are characterized by a wide heterogeneity and diversity of repeated families. These seq sequences uences can represent a large portion of the genomes and, in some cases, can exceed more than 80% of the cell DNA content [12, 13]. For a long time, no functional action was attributed to some of the repeated sequences, and these elements were known as “junk DNAs”. Although in recent years the concept of ”junk DNA” has changed mostly due to the discovery of transcribing regions of repeated elements and their involvement in genomic functions, the repeated DNAs have been defined as “encoding” and “non-encoding ” sequences. Briefly, the encoding DNAs are represented mainly by the multigenic families, such as rRNAs and histone genes, while the “non - encoding” elements are the satDNAs and transposable elements, besides the micro and minisatelites. These elements can be organized in tandem , as for example the satDNAs, or dispersed throughout the genome, as the transposable elements in general. Basically these sequences compose the nuclear genome architecture together with the less repeated sequences represented by the unique and low copy number sequences and low repeated DNAs (Fig. 2). The term “multigene family” is used to indicate groups of DNA sequences (genes) that have descended from a common ancestral gene and show notably structural similarity and function [14]. For chromosomal studies, the most used multigene families are the rRN A genes (major 45S and minor 5S transcribing units) and, in a lesser extent, the histone genes. The ribosomal DNAs (rDNA) are organized in tandem arrays with variable number of repetitions in distinct genomes. The 45S rDNA repeats contain units separated f from rom each other by intergenic spacers (IGS) and transcribes for the 18S, 5.8S and 28S rRNAs. The rRNA transcribing regions are separated from each other by internal transcribed spacers (ITS) (Fig. 3a). On the other hand, the minor 5S rRNA sequences are transcribed by the highly conserved 5S rRNA genes with 120 base pairs (bp) interspersed from each other by a non-transcribed spacer (NTS) with variable nucleotide sequence (Fig. 3b) [15]. The multigene family that codes for the histone proteins in general can be organized in one cluster formed by all intronless tandemly organized histone sequences (H1, H2a, H2b, H3 and H4), spaced by noncoding DNA sequences, as observed in the genome of Drosophila melanogaster (Figure 3c) [16]. These same genes can be distributed as single genes or group of histone genes, as in Gallus domesticus , man and mouse. Moreover, the two kinds of organization can be found in the same genome, which is the case of Xenopus laevis [17, 18]. The satDNA, minisatellite and microsatellite are tandemly arrayed and highly repeated sequences found in the eukaryotic genomes. These sequences are variable in their structure structure, , repeated unit and cluster repetition sizes. The satDNAs are organized in large clusters that are usually located in the telomeric and centromeric heterochromatic regions of the chromosomes. The size of a basic motif or repeated unit of the satDNAs can vary from 100 to 1,000 nucleotides and it can be ...