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

Abstract

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.

Role of Melt Curve Analysis in Interpretation of Nutrigenomics’ MicroRNA Expression Data

“…"uAnalyze SM " is another web-based tool, similar to uMELT, for analyzing high-resolution melting PCR products' data, in which recursive nearest neighbor thermodynamic calculations are used to predict a melt curve. Using 14 amplicons of CYBB (cytochrome b-245 heavy chain, also known as cytochromae b(558) subunit), the main±standard deviation, the difference between experimental and predicted fluorescence at 50% helicity was -0.04±0.48˚C (67).…”

“…Thus, the presence of double peaks during MCA, is not always indicative of nonspecific amplification, and other methods such as agarose gel electrophoresis, and use of melt curve prediction software (60,67) an amplicon. For example, Figure 5A shows a single peak for exon 17b of CFTE (Cystic Fibrosis Transmembrane Conductance Regulator) gene, whereas the melt curve for an amplicon from exon 7 of CFTR shows two peaks, which could be interpreted as indicative of two separate amplicons ( Figure 5B).…”

In silicosingle strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae

“…Many bioinformatic tools designed to predict melting curves of nucleic acids are available [15–17]. Softwares that predict melting curves can efficiently validate regions with different denaturation profiles and these regions can be exploited to differentiate similar sequences and to define targets to post-PCR tests [18].…”

Heterozygote PCR Product Melting Curve Prediction

“…For each of the 52 SNVs, heterozygote melting curve predictions were generated by varying the collective heteroduplex contribution from 0% to 60% by 5% increments and the ionic scaling factor for mismatches from 40% to 100% by 2% increments, resulting in 360 melting curves. The area between the predicted and experimental curves, calculated as the two‐dimensional (2D) offset [Dwight et al., ] was plotted against the collective heteroduplex percentage and the ionic scaling factor. This identified a narrower range along each axis for a high‐resolution minimization of the 2D offset from ∼1,000 in silico melting curves.…”