title: High-throughput SELEX determination of DNA sequences bound by transcription factors in vitro.
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.
authors.affiliation: Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Ogawa
authors.forename: Mark D
authors.initials: MD
authors.lastname: Biggin
mesh_terms: D004247:DNA / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D016133:Polymerase Chain Reaction; D052156:SELEX Aptamer Technique; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D014157:Transcription Factors; D004247:DNA