Sensitivity of the RCA assay in the presence of DNA mixtures. The accumulation of double-stranded DNA was detected by staining with Sybr green I. RCA signals indicative of the presence of a specific mutation are shown as exponential increases in fluorescence signal above baseline. The figure illustrates the padlock probe-RCA reaction using the Ca-Y257H-specific probe to detect varying concentrations (100%, 50%, 20%, 10% and 5%) of target template (1011copies). The target template was DNA from isolate C594 containing the Y257H mutation; this was diluted with DNA from strain ATCC 10231 (without the Y257H mutation). The intensity of RCA fluorescence signal weakened with decreased template concentration. The sensitivity of the assay corresponded to a concentration of 5% template DNA in the mixture.