Thiazole Orange as an Alternative to Antibody Binding for Detecting Triple-helical DNA in Heterochromatin of Drosophila and Rhynchosciara

The standard method for detecting triple-stranded DNA over the last 1.5 decades has been immune detection using antibodies raised against non-canonical nucleic acid structures. Many fluorescent dyes bind differentially to nucleic acids and often exhibit distinctive staining patterns along metaphase chromosomes dependent upon features, including binding to the major and minor DNA grooves, level of chromatin compaction, nucleotide specificity, and level of dye stacking. Relatively recently, the fluorochrome Thiazole Orange (TO) was shown to preferentially bind to triplex DNA in gels. Here, we demonstrate that TO also detects triplex DNA in salivary gland chromosomes of Drosophila melanogaster and Rhynchosciara americana identical in location and specificity to observations using antibodies. This finding may enable triple-stranded DNA investigations to be carried out on a much broader and reproducible scale than hitherto possible using antibodies, where a frequently encountered problem is the difference in detection specificity and sensitivity between one antibody and another.