A simple DNA Characterization method using fiber-fluorescence in situ hybridization performed without DNA fragmentation

We performed high-resolution fluorescence imaging of lambda phage DNA molecules hybridized with fluorescent-labeled DNA and peptide nucleic acid probes. In this method, the target DNA and probe were mixed, rapidly denatured and then subjected to liquid hybridization conditions. The hybridized DNA sample was then spotted onto a nontreated glass substrate and subjected to molecular combing. The resultant continuous fluorescence signal of intact lambda DNA shows that the fluorescent-labeled probes bound to the predicted sites but in a pattern that was clearly different to the beads-on-a-string pattern typical for fiber-fluorescence in situ hybridization. The key changes to the conventional method are hybridization of the free target DNA in liquid and lowering the denaturation temperature. The method described here allows the rapid and direct visualization of the specific binding sites of intact DNA molecules without damaging the DNA fibers and causing fragmentation of the fluorescence signal. This technique should be a useful tool in studies of genetics and also large-scale DNA sequencing projects.