Validation of LASR. UV melting curves (black) for DNA duplexes (a,b, and c, as indicated) and corresponding normalized single-molecule melting curves (red). Single-molecule melting curves are obtained by calculating the fraction of molecules that transition from low to high FRET after a LASR T-jump, and normalizing the high-temperature fraction to one. Error bars were calculated as , where n is the number of molecules observed.