Fluorescent in situ hybridization with transposable element probes to mitotic chromosomal heterochromatin of Drosophila
Methods in Molecular Biology 260: 29-39
ISSN/ISBN: 1064-3745 PMID: 15020800 DOI: 10.1385/1-59259-755-6:029
The technique of in situ hybridization of DNA probes to Drosophila chromosomes has been initially applied to the salivary gland polytene chromosomes and is now routinely used for mapping single-copy and repetitive DNA sequences, such as transposable elements, to the euchromatic regions of these chromosomes. However, most of the heterochromatin normally escapes cytogenetic analyses on polytene chromosomes because it is organized in a poorly differentiated cytological structure called the chromocenter. This peculiar organization does not allow a detailed mapping of DNA clones to heterochromatin. Such a limitation can be overcome by the fluorescent in situ hybridization (FISH) technique on mitotic chromosomes of D. melanogaster, where heterochromatin has been extensively characterized by banding techniques and subdivided into several cytologically diverse regions. Digital images of FISH signals and DAPI staining can be separately recorded by CCD camera, pseudocolored, and merged using specific software for image analysis. The visualization of the signals and DAPI banding pattern on a single chromosome enables the mapping of a given sequence to specific cytological regions of mitotic heterochromatin. This method has initially proven successful in the detection and mapping of transposable element clusters in the heterochromatin of D. melanogaster and has been used to study the distribution of repeated and even single-copy sequences.