Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
High-Throughput DNA Structural Analysis by Hydroxyl Radical Cleavage and Capillary Electrophoresis
DNA structure affects protein binding and rates of transcription, and directly informs cellular processes (e.g., 1). Since DNA structure depends on local base composition (2), our long-term goal, in concert with the ENCODE Project, is to identify regions within the human genome with evolutionarily conserved structure. We report here on our efforts to add to the OH Radical Cleavage Intensity Database (ORChID) (3), to improve our algorithm for predicting DNA structure, by adding cleavage data on relatively long (300-400 base pair) DNA sequences. This necessitates development of an accurate, high-throughput method for DNA shape determination.
Our lab has previously developed a method to map DNA structure using hydroxyl radical cleavage patterns (4). Hydroxyl radicals create single-stranded breaks in DNA. The frequency of damage at a particular site depends on the local structure, because of sequence-dependent variation in backbone solvent accessibility. We use this technique, coupled with the high-throughput analytical capabilities of capillary electrophoresis, to quickly evaluate the structural variation of longer DNA sequences than was previously possible using standard gel electrophoresis. Cleavage patterns at single nucleotide resolution are quantified from the fluorescence trace of the capillary electrophoresis instrument using the open-source program CAFA (capillary automated footprinting analysis) (5). This high-throughput method enables fast and accurate acquisition of DNA structural information for improvement of the ORChID algorithm.