Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, São Paulo, Brazil.
J Histochem Cytochem. 2018 Mar;66(3):143-154. doi: 10.1369/0022155417745496. Epub 2017 Dec 21.
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.
在过去的 15 年中，检测三链 DNA 的标准方法一直是使用针对非经典核酸结构的抗体进行免疫检测。许多荧光染料对核酸的结合具有差异性，并且通常在依赖于特征的中期染色体上表现出独特的染色模式，包括与主要和次要 DNA 凹槽的结合、染色质紧缩程度、核苷酸特异性和染料堆积程度。最近，荧光染料噻唑橙（TO）被证明优先与凝胶中的三链 DNA 结合。在这里，我们证明 TO 还可以检测果蝇唾液腺染色体和美洲大蠊 Rhynchosciara americana 中的三链 DNA，其位置和特异性与使用抗体的观察结果完全相同。这一发现可能使三链 DNA 的研究能够在比以前使用抗体更为广泛和可重复的范围内进行，而使用抗体时经常遇到的问题是一种抗体与另一种抗体之间的检测特异性和敏感性的差异。
