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Biological processes such as development, cell cycle and cell differentiation, are mediated through differential gene expression. These spatio-temporal differences in expression arise from modulations in gene regulatory networks, which consist of physical and functional interactions between transcription factors and their target genes. We have developed a gene-centered approach, the high-throughput gateway-compatible yeast one-hybrid (Y1H) system, which enables the identification of physical interactions between Drosophila transcription factors and regulatory elements of interest and thus allow us to generate maps describing such gene regulatory networks. While Y1H assays identify candidate interactors for DNA elements, they do not provide positional information as to where exactly these TFs bind within these DNA sequences. We therefore developed a microfluidics-based protein–DNA interaction mapping approach, termed MARE, enabling the finegrained localization of TFs of interest within specific regulatory elements. The MARE technique can be compared with a series of electrophoretic mobility shift assays, in which a TF is tested for its ability to bind to a collection of typically small DNA sequences, and relative DNA occupancy data for each sequence can be derived.