Molecular cytogenetic methodologies and a BAC probe panel resource for genomic analyses in the zebrafish

Molecular cytogenetics is a field that emerged in the 1980s, based on a technique referred to as fluorescence in situ hybridization, (FISH). Using FISH methodologies, a specific DNA sequence or collection of DNA fragments may be selectively labeled with a hapten molecule or fluorescent dye and hybridized to denatured chromosomes, interphase cells, or even chromatin fibers. DNA hybridization kinetics permit these labeled probes to anneal to their complementary sequences on such chromosomal DNA preparations allowing for direct visualization of the sequence of interest in the genome being interrogated. If present, the relative chromosomal position of the sequence can sometimes also be ascertained. Progress in molecular cytogenetic research has advanced the genetic characterization of zebrafish models of human diseases as well as assisted with accurate annotation of the zebrafish reference genome by anchoring large DNA fragments to specific chromosome regions. Using the procedures described in this chapter, hundreds of ambiguous zebrafish bacterial artificial chromosome (BAC) clones have already been assigned to individual genetic linkage groups. Molecular cytogenetic techniques can also be used to study gene duplication events and study the molecular mechanisms by which they arise. Moreover, the availability of a new molecular cytogenetic technique, array-based comparative genomic hybridization (aCGH), is now able to identify gains and losses of DNA segments in zebrafish DNA samples in a genome-wide manner and in a single assay.