1. Outline of the chromatin immunoprecipitation (ChIP) technique
Outline of the chromatin immunoprecipitation (ChIP) technique. This method allows for the precise localization of a particular protein (or modified protein if an appropriate antibody is available; eg, phosphorylated or acetylated histones, transcription factors, etc) on a particular sequence element in living cells. Depending upon the method used to analyze the immunopurified DNA, quantitative or semiquantitative information, at near nucleotide level resolution, can be obtained. Protein-DNA occupancy can be scored genome-wide in two ways. First, by ChIP-chip, a method that uses a hybridization readout. In ChIP-chip total genomic DNA is labeled with one particular fluorophore and the immunopurified DNA is labeled with a spectrally distinct fluorophore. These differentially labeled DNAs are mixed and hybridized to microarray “chips” (microscope slides) that contain specific DNA fragments, or more commonly now, synthetic oligonucleotide 50 to 70 nucleotides long. These gene-specific oligonucleotides are deposited and covalently attached at predetermined, known X,Y position/coordinates on the slide. The labeled DNAs are hybridized, the slides washed and hybridization to each gene-specific oligonucleotide probe is scored using differential laser scanning and sensitive photodetection at micron resolution. The hybridization signal intensities are quantified and the ratio of IP DNA/genomic DNA signals is used to score occupancy levels. The second method, termed ChIP-Seq, directly sequences immunopurified DNAs using NGS sequencing methods. Two variants of ChIP-Seq are shown: “standard” ChIP-Seq and ChIP-Exo. These two approaches differ in their ability to resolve and map the locations of the bound protein on genomic DNA. Standard ChIP-Seq resolution is ∼ ±50 nt resolution, while ChIP-Exo has near single nt level resolution. Both approaches rely upon efficient bioinformatic algorithms to deal with the very large datasets that are generated. ChIP-chip and ChIP-Seq techniques provide a (semi-) quantitative measure of in vivo protein occupancy. Though not schematized here, similar methods termed RIP (RNA immunoprecipitation) or CLIP (crosslinking protein-RNA and immunoprecipitation), which differ primarily in the method of protein-RNA crosslinking, can score the in vivo binding of specific proteins to specific RNA species (typically mRNAs, though any RNA species can be analyzed by these techniques).
Source: Molecular Genetics, Recombinant DNA, & Genomic Technology, Harper's Illustrated Biochemistry, 30e
Citation: Rodwell VW, Bender DA, Botham KM, Kennelly PJ, Weil P. Harper's Illustrated Biochemistry, 30e; 2015 Available at: Accessed: October 10, 2017
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