Abstract
Protein-binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA-binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding-site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF-binding specificities at high resolution using such 'all 10-mer' universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day.
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Acknowledgements
We thank Anthony Philippakis for helpful discussion, Andrew Gehrke for technical assistance and Manuel Llinas and Stephen Gisselbrecht for helpful comments and critical reading of the manuscript. M.F.B. and M.L.B. were funded by NIH/NHGRI grant no. R01 HG003985.
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M.L. Bulyk is a co-inventor on United States Patent 6,548,021 entitled “Surface-bound, double-stranded DNA protein arrays”, and is also a co-inventor on a pending patent on the sequence design of the universal arrays utilized in this paper.
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Berger, M., Bulyk, M. Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors. Nat Protoc 4, 393–411 (2009). https://doi.org/10.1038/nprot.2008.195
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DOI: https://doi.org/10.1038/nprot.2008.195
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