Cell type-specific profiling of protein-DNA interactions without cell isolation using Targeted DamID with next-generation sequencing

By Owen J. Marshall, Tony D. Southall, Andrea H. Brand. Nature Protocols, 2007

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Understanding gene regulatory networks in mammalian cells requires detailed knowledge of protein–DNA interactions. Commonly used methods for genome-wide mapping of these interactions are based on chromatin immunoprecipitation. However, these methods have some drawbacks, such as the use of crosslinking reagents, the need for highly specific antibodies and relatively large amounts of starting material. We present DamID, an alternative technique to map genome-wide occupancy of interaction sites in vivo, that bypasses these limitations. DamID is based on the expression of a fusion protein consisting of a protein of interest and DNA adenine methyltransferase (Dam). This leads to methylation of adenines near sites where the protein of interest interacts with the DNA. These methylated sequences are subsequently amplified by a methylation-specific PCR protocol and identified by hybridization to microarrays. Using DamID, genome-wide maps of the binding of DNA-interacting proteins in mammalian cells can be constructed efficiently. Depending on the strategy used for expression of the Dam-fusion proteins, genome-wide binding maps can be obtained in as little as 2 weeks.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/ff12a133527d8b98d376a3eafaa2b5ec961b3eb3

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