Abstract
SELEX (systematic evolution of ligands by exponential enrichment) was created 20 years ago as a method to enrich small populations of bound DNAs from a random sequence pool by PCR amplification. It provides a powerful way to determine the in vitro binding specificities of DNA-binding proteins such as transcription factors. Here, we present a robust version of the SELEX protocol for high-throughput analysis. Protein-bound beads prepared from insoluble recombinant 6× HIS-tagged transcription factor protein are used in a simple pull-down assay. To allow efficient determination of the enriched DNA sequences, bound oligonucleotides are concatenated, allowing approximately 1,000 oligonucleotides to be sequenced from one 96-well format plate. Successive rounds of SELEX data are statistically useful for understanding the full range of moderate affinity and high-affinity binding sites.
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Reference URLs
SAGE protocol: http://www.sagenet.org/protocol/index.htm
Acknowledgments
The authors would like to thank Michael B. Eisen, David Nix, Stuart Davidson, Juli Atherton, Nathan Boley, Ben Brown, and Peter Bickel for SELEX data analysis. Dr. Mark Stapleton for cloning of recombinant transcription factor genes, and Lucy Zeng for performing many SELEX experiments. This work was supported by the US National Institutes of Health (NIH) under grant GM704403. Work at Lawrence Berkeley National Laboratory was conducted under Department of Energy contract DE-AC02-05CH11231.
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Ogawa, N., Biggin, M.D. (2012). High-Throughput SELEX Determination of DNA Sequences Bound by Transcription Factors In Vitro. In: Deplancke, B., Gheldof, N. (eds) Gene Regulatory Networks. Methods in Molecular Biology, vol 786. Humana Press. https://doi.org/10.1007/978-1-61779-292-2_3
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DOI: https://doi.org/10.1007/978-1-61779-292-2_3
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