Details

Autor(en) / Beteiligte

• Ogawa, Nobuo
• Biggin, Mark D

Titel

High-throughput SELEX determination of DNA sequences bound by transcription factors in vitro

Ist Teil von

• Methods in molecular biology (Clifton, N.J.), 2012, Vol.786, p.51

Ort / Verlag

United States

Erscheinungsjahr

2012

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.