uAnalyze: Web-Based High-Resolution DNA Melting Analysis with Comparison to Thermodynamic Predictions

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 mel...

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Bibliographic Details
Published in:IEEE/ACM transactions on computational biology and bioinformatics 2012-11, Vol.9 (6), p.1805-1811
Main Authors: Dwight, Z. L., Palais, R., Wittwer, C. T.
Format: Article
Language:English
Subjects:
Bioinformatics
biology and genetics
Computational biology
Computational Biology - methods
DNA - analysis
DNA - chemistry
high-resolution melting
Humans
Internet
Melting curve analysis
Membrane Glycoproteins - genetics
modeling and prediction
Models, Genetic
Models, Statistical
NADPH Oxidase 2
NADPH Oxidases - genetics
Nucleic Acid Denaturation
Polymerase Chain Reaction - methods
Software
Thermodynamics
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Description
Summary: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.
ISSN:1545-5963
1557-9964
DOI:10.1109/TCBB.2012.112