FIG. 2.

Determination of the stability of probe-target hybrids formed between sloppy molecular beacon probe A and seven different mycobacterial target oligonucleotides. (A) The fluorescence intensity of the molecular beacon in each hybrid was measured as a function of temperature. A drop in fluorescence intensity occurs at those temperatures where each hybrid dissociates. The increase in fluorescence intensity observed at higher temperatures with each species, and with the no-target control (black line), is due to the melting apart of the probe's hairpin stem. Each of the seven target oligonucleotides formed hybrids of differing stability (from weakest to strongest: M. aichiense, M. diernhoferi, M. abscessus, M. lentiflavum, M. senegalense, M. terrae, and M. asiaticum). (B) Utilizing the data from the left-hand panel, the derivative of fluorescence intensity with respect to temperature (dI/dT) is plotted as a function of temperature. (C) The data within approximately 2°C of each peak in the middle panel were normalized to the same height.