title: Semiautomated and rapid quantification of nucleic acid footprinting and structure mapping experiments.
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 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.
authors.affiliation: Department of Developmental Genetics and Bioinformatics, Wadsworth Center, Albany, New York 12208, USA. alain@wadsworth.org
authors.forename: Alain
authors.initials: A
authors.lastname: Laederach
authors.forename: Rhiju
authors.initials: R
authors.lastname: Das
authors.forename: Quentin
authors.initials: Q
authors.lastname: Vicens
authors.forename: Samuel M
authors.initials: SM
authors.lastname: Pearlman
authors.forename: Michael
authors.initials: M
authors.lastname: Brenowitz
authors.forename: Daniel
authors.initials: D
authors.lastname: Herschlag
authors.forename: Russ B
authors.initials: RB
authors.lastname: Altman
mesh_terms: D018983:DNA Footprinting / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D009690:Nucleic Acid Conformation; D012984:Software
references.citation: Biochemistry. 2000 Oct 10;39(40):12465-75
references.pmid: 11015228
references.citation: Nat Protoc. 2006;1(3):1610-6
references.pmid: 17406453
references.citation: J Am Chem Soc. 2005 Apr 6;127(13):4659-67
references.pmid: 15796531
references.citation: Biochemistry. 2000 Sep 12;39(36):10975-85
references.pmid: 10998234
references.citation: RNA. 2006 Apr;12(4):533-41
references.pmid: 16484377
references.citation: Proc Natl Acad Sci U S A. 2007 Apr 24;104(17):7045-50
references.pmid: 17438287
references.citation: J Mol Biol. 2004 Nov 5;343(5):1195-206
references.pmid: 15491606
references.citation: Science. 1989 Jul 21;245(4915):276-82
references.pmid: 2501870
references.citation: J Mol Biol. 2003 Sep 12;332(2):311-9
references.pmid: 12948483
references.citation: Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):1113-6
references.pmid: 7862644
references.citation: J Mol Biol. 2006 May 12;358(4):1179-90
references.pmid: 16574145
references.citation: RNA. 2007 Apr;13(4):536-48
references.pmid: 17299128
references.citation: Nucleic Acids Res. 1985 Dec 9;13(23):8339-57
references.pmid: 2417197
references.citation: J Mol Biol. 2003 May 16;328(5):1011-26
references.pmid: 12729738
references.citation: J Mol Biol. 2005 Nov 25;354(2):483-96
references.pmid: 16242711
references.citation: Biochemistry. 2002 May 7;41(18):5799-806
references.pmid: 11980483
references.citation: RNA. 2005 Mar;11(3):344-54
references.pmid: 15701734
references.citation: J Mol Biol. 1997 Feb 14;266(1):144-59
references.pmid: 9054977
references.citation: Nucleic Acids Res. 2004 Aug 19;32(15):E119
references.pmid: 15319447
references.citation: Nat Protoc. 2007;2(10):2608-23
references.pmid: 17948004
references.citation: Proc Natl Acad Sci U S A. 1985 Feb;82(3):648-52
references.pmid: 2579378
references.citation: Proc Natl Acad Sci U S A. 1986 Nov;83(22):8462-6
references.pmid: 3464963
references.citation: Nucleic Acids Res. 2006 Mar 31;34(6):e48
references.pmid: 16582097
references.citation: J Mol Biol. 2001 May 18;308(5):839-51
references.pmid: 11352576
references.citation: Nat Chem Biol. 2005 Jul;1(2):104-11
references.pmid: 16408007
references.citation: Science. 1998 Mar 20;279(5358):1940-3
references.pmid: 9506944