Das Rhiju, Laederach Alain, Pearlman Samuel M, Herschlag Daniel, Altman Russ B
Department of Physics, Stanford University, Stanford, CA 94305,USA.
RNA. 2005 Mar;11(3):344-54. doi: 10.1261/rna.7214405.
Footprinting is a powerful and widely used tool for characterizing the structure, thermodynamics, and kinetics of nucleic acid folding and ligand binding reactions. However, quantitative analysis of the gel images produced by footprinting experiments is tedious and time-consuming, due to the absence of informatics tools specifically designed for footprinting analysis. We have developed SAFA, a semi-automated footprinting analysis software package that achieves accurate gel quantification while reducing the time to analyze a gel from several hours to 15 min or less. The increase in analysis speed is achieved through a graphical user interface that implements a novel methodology for lane and band assignment, called "gel rectification," and an optimized band deconvolution algorithm. The SAFA software yields results that are consistent with published methodologies and reduces the investigator-dependent variability compared to less automated methods. These software developments simplify the analysis procedure for a footprinting gel and can therefore facilitate the use of quantitative footprinting techniques in nucleic acid laboratories that otherwise might not have considered their use. Further, the increased throughput provided by SAFA may allow a more comprehensive understanding of molecular interactions. The software and documentation are freely available for download at http://safa.stanford.edu.
足迹法是一种功能强大且广泛应用的工具，用于表征核酸折叠以及配体结合反应的结构、热力学和动力学。然而，由于缺乏专门为足迹分析设计的信息学工具，对足迹实验产生的凝胶图像进行定量分析既繁琐又耗时。我们开发了SAFA，这是一个半自动足迹分析软件包，它能实现准确的凝胶定量，同时将分析凝胶的时间从数小时减少到15分钟或更短。分析速度的提高是通过一个图形用户界面实现的，该界面采用了一种名为“凝胶校正”的用于泳道和条带分配的新方法，以及一种优化的条带反卷积算法。SAFA软件产生的结果与已发表的方法一致，并且与自动化程度较低的方法相比，减少了因研究者而异的变异性。这些软件开发简化了足迹凝胶的分析过程，因此可以促进核酸实验室中定量足迹技术的使用，否则这些实验室可能不会考虑使用该技术。此外，SAFA提供的更高通量可能会让人对分子相互作用有更全面的理解。该软件和文档可在http://safa.stanford.edu免费下载。
