title: Simple method for fluorescence DNA in situ hybridization to squashed chromosomes.
abstract: DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.
authors.affiliation: Department of Biology, University of Rochester; alarracu@bio.rochester.edu.
authors.forename: Amanda M
authors.initials: AM
authors.lastname: Larracuente
authors.affiliation: W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges; pferree@kecksci.claremont.edu.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Ferree
mesh_terms: D000818:Animals; D002875:Chromosomes / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry; D004331:Drosophila melanogaster; D005456:Fluorescent Dyes / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D012091:Repetitive Sequences, Nucleic Acid; D014863:Wasps
chemical_list: D015342:DNA Probes; D005456:Fluorescent Dyes; D004247:DNA