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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 is a modification of the ChIP-seq protocol, improving the resolution of binding sites from hundreds of base pairs to almost one base pair. 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. The nucleotides of the exonuclease-treated ends are determined using some combination of DNA sequencing, microarrays, and PCR. These sequences are then mapped to the genome to identify the locations on the genome at which the protein binds.
==Theory==
Chromatin immunoprecipitation (ChIP) techniques have been in use since 1984 to detect protein-DNA interactions. There have been many variations on ChIP to improve the quality of results. One such improvement, ChIP-on-chip (ChIP-chip), combines ChIP with microarray technology. 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. Next came ChIP-sequencing (ChIP-seq), which combines ChIP with high-throughput sequencing. However, the heterogeneous nature of sheared DNA fragments maps binding sites to within ±300 base pairs, limiting specificity. Secondly, contaminating DNA presents a grave problem since so few genetic loci are cross-linked to the protein of interest, making any non-specific genomic DNA a significant source of background noise.
To address these problems, Rhee and Pugh revised the classic nuclease protection assay to develop ChIP-exo. This new ChIP technique relies on a lambda exonuclease that degrades only, and all, unbound double-stranded DNA in the 5′-3′ direction. 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. Cells are then collected, broken open, and the chromatin sheared and solubilized by sonication. An antibody is then used to immunoprecipitate the protein of interest, along with the crosslinked DNA. DNA PCR adaptors are then ligated to the ends, which serve as a priming point for second strand DNA synthesis after the exonuclease digestion. Lambda exonuclease then digests double DNA strands from the 5′ end until digestion is blocked at the border of the protein-DNA covalent interaction. Most contaminating DNA is degraded by the addition of a second single-strand specific exonuclease. After the cross-linking is reversed, the primers to the PCR adaptors are extended to form double stranded DNA, and a second adaptor is ligated to 5′ ends to demarcate the precise location of exonuclease digestion cessation. The library is then amplified by PCR, and the products are identified by high throughput sequencing. This method allows for resolution of up to a single base pair for any protein binding site within any genome, which is a much higher resolution than either ChIP-chip or ChIP-seq.
抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』
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