ChIP-exo

ChIP-exo is a chromatin immunoprecipitation based method for mapping the locations at which a protein of interest (transcription factor) binds to the genome.

It employs the use of exonucleases to degrade strands of the protein-bound DNA in the 5'-3' direction to within a small number of nucleotides of the protein binding site.

This technique has limited sensitivity and specificity, especially in vivo where microarrays are constrained by thousands of proteins present in the nuclear compartment, resulting in a high rate of false positives.

[3] However, the heterogeneous nature of sheared DNA fragments maps binding sites to within ±300 base pairs, limiting specificity.

Briefly, a protein of interest (engineering one with an epitope tag can be useful for immunoprecipitation) is crosslinked in vivo to its natural binding locations across a genome using formaldehyde.

ChIP-exo has been shown to give up to single base pair resolution in identifying protein binding locations.

[4] Contamination of non-protein-bound DNA fragments can result in a high rate of false positives and negatives in ChIP experiments.

The addition of exonucleases to the process not only improves resolution of binding-site calling, but removes contaminating DNA from the solution before sequencing.

Rhee and Pugh introduce ChIP-exo by performing analyses on a small collection of transcription factors: Reb1, Gal4, Phd1, Rap1 in yeast and CTCF in human.