Semiautomated and rapid quantification of nucleic acid footprinting and structure mapping experiments.
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 approximately 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.
Laederach
Alain
A
Department of Developmental Genetics and Bioinformatics, Wadsworth Center, Albany, New York 12208, USA. alain@wadsworth.org
Das
Rhiju
R
Vicens
Quentin
Q
Pearlman
Samuel M
SM
Brenowitz
Michael
M
Herschlag
Daniel
D
Altman
Russ B
RB
D018983:DNA Footprinting / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D009690:Nucleic Acid Conformation; D012984:Software
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