Nucleic Acids Research Advance Access published online on October 20, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp802
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Methods Online |
Bind-n-Seq: high-throughput analysis of in vitro protein–DNA interactions using massively parallel sequencing
Genome Center, University of California, Davis, CA 95616, USA
*To whom correspondence should be addressed. Tel: +1 530 754 9134; Fax: +1 530 754 9658; Email: djsegal{at}ucdavis.edu Correspondence may also be addressed to Ian Korf. Tel: +1 530 754 4989; Fax: +1 530 754 9658; Email: ifkorf{at}ucdavis.edu
Received June 16, 2009. Revised August 3, 2009. Accepted September 14, 2009.
Transcription factor–DNA interactions are some of the most important processes in biology because they directly control hereditary information. The targets of most transcription factor are unknown. In this report, we introduce Bind-n-Seq, a new high-throughput method for analyzing protein–DNA interactions in vitro, with several advantages over current methods. The procedure has three steps (i) binding proteins to randomized oligonucleotide DNA targets, (ii) sequencing the bound oligonucleotide with massively parallel technology and (iii) finding motifs among the sequences. De novo binding motifs determined by this method for the DNA-binding domains of two well-characterized zinc-finger proteins were similar to those described previously. Furthermore, calculations of the relative affinity of the proteins for specific DNA sequences correlated significantly with previous studies (R2 = 0.9). These results present Bind-n-Seq as a highly rapid and parallel method for determining in vitro binding sites and relative affinities.