Simbios Talk by Alain Laederach, Stanford University, January 23, 2008

Title:  High-throughput single-nucleotide structural mapping by CAFA (Capillary Automated Footprinting Analysis)

The use of capillary electrophoresis with fluorescently labeled nucleic acids has revolutionized the field of DNA sequencing, effectively fueling the genomic revolution. We present an alternative use of this technology for the high-throughput structural analysis of nucleic acids by chemical and/or biochemical mapping (‘footprinting’). Combining direct and indirect fluorophore labeling of nucleic acids with novel quantitation algorithms, we achieve the high throughput and data quality necessary for genomic-scale structural analysis. The CAFA (Capillary Automated Footprinting Analysis) software that we have developed is available gratis as an open-source application that can be downloaded from The accuracy, throughput, and reproducibility of quantitative capillary electrophoretic analysis are demonstrated using hydroxyl radical analysis of RNA. The versatility of CAFA is illustrated by dimethyl sulfate analysis of RNA secondary structure and DNase I mapping of a protein binding to a specific sequence of DNA. We carefully evaluate the experimental characteristics of the method and developed the computational tools necessary to ensure accurate and reproducible data. For example, the primer extension approach to indirectly end label reaction products is subject to intrinsic stops that result in peaks unrelated to the experiment.  We developed an algorithm that uses a single background sample to predict peaks of low reproducibility that are then excluded from analysis. We demonstrate robust quantification with high precision that minimizes the need for replicate experiments.