ChIP-Sequencing, also known as ChIP-Seq, is the next frontier of technology used to analyze protein interactions with DNA. ChIP-Seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify binding sites of DNA-associated proteins. It can be used to precisely and cost-effectively map global binding sites for any protein of interest. Previously, ChIP-on-chip was the most common technique utilized to study these protein-DNA relations.
Specific DNA sites in direct physical interaction with transcription factors and other proteins can be isolated by chromatin immunoprecipitation. ChIP produces a library of target DNA sites bound to a target in vivo. Massively parallel sequence analysis are used in conjunction with whole-genome sequence databases to analyze the interaction pattern of any protein with DNA, or the pattern of any epigenetic chromatin modifications. This can be applied to the set of ChIP-able proteins and modifications, such as transcription factors, polymerases and transcriptional machinery, structural proteins, protein modifications, and DNA modifications.
There are many new sequencing methods used in this sequencing step. Some technologies that analyze the sequences can use cluster amplification of adapter-ligated ChIP DNA fragments on a solid flow cell substrate to create clusters of approximately 1000 clonal copies each. The resulting high density array of template clusters on the flow cell surface is sequenced by a Genome analyzing program. Each template cluster undergoes sequencing-by-synthesis in parallel using novel fluorescently labelled reversible terminator nucleotides. Templates are sequenced base-by-base during each read. Then, the data collection and analysis software aligns sample sequences to a known genomic sequence to identify the ChIP-DNA fragments.
Unlike microarray-based ChIP methods, the accuracy of the ChIP-Seq assay is not limited by the spacing of predetermined probes. By integrating a large number of short reads, highly precise binding site localization is obtained. ChIP-Seq can locate the protein binding site within 50 base pairs. Binding affinities of a protein to different DNA sites can also be compared by quantifying the number of appearances of a given sequence.
|Starting Material||Low, > 10ng||> 4ug||Less DNA needed|
|Flexibility||Genome-wide assay available||Limited||Not limited by micro-array content|
|Positional resolution||+/- 50bp||+/- 500-1000bp||More precise site mapping|
|Sensitivity||High||Moderate||Increased reads increases sensitivity|
|Cross-hybridization||None: Each DNA is individually sequenced||Significant||Produces higher quality data|
In summary, ChIP-seq offers important advantages over ChIP-chip, including lower cost, minimal hands-on processing and a requirement for fewer replicate experiments as well as less input material. Moreover, the Stat1 experimental ChIP-seq data have a high degree of similarity to results obtained by ChIP-chip for the same type of experiment, with >64% of peaks in shared genomic regions. Because the data are sequence reads, ChIP-seq offers a rapid analysis pipeline (as long as a high-quality genome sequence is available for read mapping) as well as the potential to detect mutations in binding-site sequences, which may directly support any observed changes in protein binding and gene regulation.