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Abstract for Mapping Intracellular Protein:DNA Interactions: A
more robust and efficient alternative to ChIP (Chromatin
Immunoprecipitation)
Danette D. Hartzell, Marjeta Urh, Natasha Karassina, Georgyi Los,
and Keith Wood
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711
Regulation of chromatin structure and gene expression is
essential for normal development and cellular growth.
Transcriptional events are tightly controlled both spatially and
temporally by specific protein:DNA interactions. While significant advances in DNA tiling
and microarrays have allowed for genome-wide screening of chromatin
recognition sites, current methods to isolate intracellular
protein:DNA complexes remain cumbersome and require
coimmunoprecipitation, a process inherently subject to capture of
nonspecific DNA and proteins. To address these concerns a novel
method has been devised for the covalent capture of protein:DNA
complexes that does not require the use of antibodies. Proteins of
interest are expressed in cells as HaloTag® fusion proteins,
crosslinked to DNA, and then captured on HaloLink™ resin, which
forms a highly specific, covalent interaction with HaloTag®. Due to
the complete covalent linkage established between the resin and the
crosslinked protein:DNA complexes, the resin can be stringently
washed to remove non-specific DNA and proteins much more effectively
than is possible by co-immunoprecipitation. The crosslinks are
reversed to release purified DNA fragments from the resin. By
improving specificity and reducing background interference during
the isolation of protein:DNA complexes, this new methodology
effectively increases the signal-to-noise ratio to permit detection
of small changes in protein binding within a genome. |
