uAnalyze(SM) is a web-based tool for analyzing high-resolution melting data of PCR products. PCR product sequence is input by the user and recursive nearest neighbor thermodynamic calculations used to predict a melting curve similar to uMELT(http://www.dna.utah.edu/umelt/umelt.html). Unprocessed melting data are input directly from LightScanner-96, LS32, or HR-1 data files or via a generic format for other instruments. A fluorescence discriminator identifies low intensity samples to prevent analysis of data that cannot be adequately normalized. Temperature regions that define fluorescence background are initialized by prediction and optionally adjusted by the user. Background is removed either as an exponential or by linear baseline extrapolation. The precision or, “curve spread,” of experimental melting curves is quantified as the average of the maximum helicity difference of all curve pairs. Melting curve accuracy is quantified as the area or “2D offset” between the average experimental and predicted melting curves. Optional temperature overlay (temperature shifting) is provided to focus on curve shape. Using 14 amplicons of CYBB, the mean + / - standard deviation of the difference between experimental and predicted fluorescence at 50 percent helicity was 0:04 + / - 0:48°C. uAnalyze requires Flash, is not browser specific and can be accessed at http://www.dna.utah.edu/uv/uanalyze.html.

UNLABELLED: uMelt(SM) is a flexible web-based tool for predicting DNA melting curves and denaturation profiles of PCR products. The user defines an amplicon sequence and chooses a set of thermodynamic and experimental parameters that include nearest neighbor stacking energies, loop entropy effects, cation (monovalent and Mg(++)) concentrations and a temperature range. Using an accelerated partition function algorithm along with chosen parameter values, uMelt interactively calculates and visualizes the mean helicity and the dissociation probability at each sequence position at temperatures within the temperature range. Predicted curves display the mean helicity as a function of temperature or as derivative plots. Predicted profiles display stability as a function of sequence position either as 50% helicity temperatures or as the helicity probability at specific temperatures. The loss of helicity associated with increasing temperature may be viewed dynamically to visualize domain formation within the molecule. Results from fluorescent high-resolution melting experiments match the number of predicted melting domains and their relative temperatures. However, the absolute melting temperatures vary with the selected thermodynamic parameters and current libraries do not account for the rapid melting rates and helix stabilizing dyes used in fluorescent melting experiments. uMelt provides a convenient platform for simulation and design of high-resolution melting assays. AVAILABILITY AND IMPLEMENTATION: The application was developed in Actionscript and can be found online at http://www.dna.utah.edu/umelt/umelt.html. Adobe Flash is required to run in all browsers.