uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application
uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application
复制标题
DOI：
10.1093/bioinformatics/btr065
复制
发表时间：
2011-04-01
期刊：
影响因子：
5.8
通讯作者：
Wittwer, Carl T.
中科院分区：
文献类型：
--
作者：
Dwight, Zachary;Palais, Robert;Wittwer, Carl T.
uMelt（SM）是一个灵活的基于网络的工具，用于预测DNA融化曲线和PCR产物的变性谱。用户定义一个扩增子序列，并选择一组热力学和实验参数，包括最近邻堆叠能，环路熵效应，阳离子（一价和Mg(++)）浓度和温度范围。uMelt使用加速配分函数算法和选定的参数值，交互式地计算和可视化温度范围内温度下每个序列位置的平均螺旋度和解离概率。预测曲线显示平均螺旋度作为温度的函数或作为导数图。预测剖面的稳定性表现为序列位置在50%螺旋度温度或特定温度下的螺旋度概率的函数。随着温度的升高，螺旋度的损失可以动态地观察，以可视化分子内结构域的形成。荧光高分辨率熔化实验的结果与预测的熔化区数目及其相对温度相符。然而，绝对熔融温度随所选择的热力学参数而变化，并且目前的文库没有考虑到荧光熔融实验中使用的快速熔化速率和螺旋稳定染料。uMelt为模拟和设计高分辨率熔融试验提供了方便的平台。
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.