Identification of non-specific hybridization using an empirical equation fitted to non-equilibrium dissociation curves

Non-equilibrium dissociation curves (NEDCs) have the potential to identify non-specific hybridizations on high throughput, diagnostic microarrays. We report a simple method for the identification of non-specific signals by using a new parameter that does not rely on comparison of perfect match and mismatch dissociations. The parameter is the ratio of specific dissociation temperature (T(d-w)) to theoretical melting temperature (T(m)) and can be obtained by automated fitting of a four-parameter, sigmoid, empirical equation to the thousands of curves generated in a typical experiment. The curves fit perfect match NEDCs from an initial experiment with an R(2) of 0.998±0.006 and root mean square of 108±91 fluorescent units. Receiver operating characteristic curve analysis showed low temperature hybridization signals (20-48°C) to be as effective as area under the curve as primary data filters. Evaluation of three datasets that target 16S rRNA and functional genes with varying degrees of target sequence similarity showed that filtering out hybridizations with T(d-w)/T(m)<0.78 greatly reduced false positive results. In conclusion, T(d-w)/T(m) successfully screened many non-specific hybridizations that could not be identified using single temperature signal intensities alone, while the empirical modeling allowed a simplified approach to the high throughput analysis of thousands of NEDCs.