Protocol abstract
Nature Protocols 3, - 1395 - 1401 (2008)
Published online: 7 August 2008 | doi:10.1038/nprot.2008.134
Subject Categories: Computational and theoretical biology | Genetic analysis
Semiautomated and rapid quantification of nucleic acid footprinting and structure mapping experiments
Alain Laederach1,2,3, Rhiju Das4, Quentin Vicens5, Samuel M Pearlman6, Michael Brenowitz7, Daniel Herschlag8 & Russ B Altman2,3
Abstract
We have developed protocols for rapidly quantifying the band intensities from nucleic acid chemical mapping gels at single-nucleotide resolution. These protocols are implemented in the software SAFA (semi-automated footprinting analysis) that can be downloaded without charge from http://safa.stanford.edu. The protocols implemented in SAFA have five steps: (i) lane identification, (ii) gel rectification, (iii) band assignment, (iv) model fitting and (v) band-intensity normalization. SAFA enables the rapid quantitation of gel images containing thousands of discrete bands, thereby eliminating a bottleneck to the analysis of chemical mapping experiments. An experienced user of the software can quantify a gel image in 
20 min. Although SAFA was developed to analyze hydroxyl radical (
OH) footprints, it effectively quantifies the gel images obtained with other types of chemical mapping probes. We also present a series of tutorial movies that illustrate the best practices and different steps in the SAFA analysis as a supplement to this protocol.
- Department of Developmental Genetics and Bioinformatics, Wadsworth Center, Albany, New York 12208, USA.
- Department of Bioengineering, Stanford University, Stanford, California 94305, USA.
- Department of Genetics, Stanford University, Stanford, California 94305, USA.
- Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
- Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
- Biomedical Informatics Program, Stanford University, Stanford, California 94304, USA.
- Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
- Department of Biochemistry, Stanford University, Stanford, California 94305, USA.
Correspondence to: Alain Laederach1,2,3 e-mail: alain@wadsworth.org
