Observed distributions of the two investigated measures of target-sequence secondary structure are shown, plotted against the three categories of assay functionality (quality patterns 1, 2, and 3) for 89 crudely designed DASH assays. (A) experimentally determined T m values. (B) theoretically predicted (mfold at 37°C) ΔG values. Each data point represents the respective measure of target secondary-structure stability for one assay. Mean values are denoted by short horizontal lines. It is apparent that the mean values for failed assays (pattern 3) were significantly higher than for functional assays (patterns 1 and 2), indicating that target secondary structure is a primary cause of assay failure.
