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| title | chunk | source | category | tags | date_saved | instance |
|---|---|---|---|---|---|---|
| ChIP-on-chip | 3/3 | https://en.wikipedia.org/wiki/ChIP-on-chip | reference | science, encyclopedia | 2026-05-05T14:02:11.302460+00:00 | kb-cron |
== Strengths and weaknesses == Using tiled arrays, ChIP-on-chip allows for high resolution of genome-wide maps. These maps can determine the binding sites of many DNA-binding proteins like transcription factors and also chromatin modifications. Although ChIP-on-chip can be a powerful technique in the area of genomics, it is very expensive. Most published studies using ChIP-on-chip repeat their experiments at least three times to ensure biologically meaningful maps. The cost of the DNA microarrays is often a limiting factor to whether a laboratory should proceed with a ChIP-on-chip experiment. Another limitation is the size of DNA fragments that can be achieved. Most ChIP-on-chip protocols utilize sonication as a method of breaking up DNA into small pieces. However, sonication is limited to a minimal fragment size of 200 bp. For higher resolution maps, this limitation should be overcome to achieve smaller fragments, preferably to single nucleosome resolution. As mentioned previously, the statistical analysis of the huge amount of data generated from arrays is a challenge and normalization procedures should aim to minimize artifacts and determine what is really biologically significant. So far, application to mammalian genomes has been a major limitation, for example, due to the significant percentage of the genome that is occupied by repeats. However, as ChIP-on-chip technology advances, high resolution whole mammalian genome maps should become achievable. Antibodies used for ChIP-on-chip can be an important limiting factor. ChIP-on-chip requires highly specific antibodies that must recognize its epitope in free solution and also under fixed conditions. If it is demonstrated to successfully immunoprecipitate cross-linked chromatin, it is termed "ChIP-grade". Companies that provide ChIP-grade antibodies include Abcam, Cell Signaling Technology, Santa Cruz, and Upstate. To overcome the problem of specificity, the protein of interest can be fused to a tag like FLAG or HA that are recognized by antibodies. An alternative to ChIP-on-chip that does not require antibodies is DamID. Also available are antibodies against a specific histone modification like H3 tri methyl K4. As mentioned before, the combination of these antibodies and ChIP-on-chip has become extremely powerful in determining whole genome analysis of histone modification patterns and will contribute tremendously to our understanding of the histone code and epigenetics. A study demonstrating the non-specific nature of DNA binding proteins has been published in PLoS Biology. This indicates that alternate confirmation of functional relevancy is a necessary step in any ChIP-chip experiment.
== History == A first ChIP-on-chip experiment was performed in 1999 to analyze the distribution of cohesin along budding yeast chromosome III. Although the genome was not completely represented, the protocol in this study remains equivalent as those used in later studies. The ChIP-on-chip technique using all of the ORFs of the genome (that nevertheless remains incomplete, missing intergenic regions) was then applied successfully in three papers published in 2000 and 2001. The authors identified binding sites for individual transcription factors in the budding yeast Saccharomyces cerevisiae. In 2002, Richard Young's group determined the genome-wide positions of 106 transcription factors using a c-Myc tagging system in yeast. The first demonstration of the mammalian ChIp-on-chip technique reported the isolation of nine chromatin fragments containing weak and strong E2F binding site was done by Peggy Farnham's lab in collaboration with Michael Zhang's lab and published in 2001. This study was followed several months later in a collaboration between the Young lab with the laboratory of Brian Dynlacht which used the ChIP-on-chip technique to show for the first time that E2F targets encode components of the DNA damage checkpoint and repair pathways, as well as factors involved in chromatin assembly/condensation, chromosome segregation, and the mitotic spindle checkpoint Other applications for ChIP-on-chip include DNA replication, recombination, and chromatin structure. Since then, ChIP-on-chip has become a powerful tool in determining genome-wide maps of histone modifications and many more transcription factors. ChIP-on-chip in mammalian systems has been difficult due to the large and repetitive genomes. Thus, many studies in mammalian cells have focused on select promoter regions that are predicted to bind transcription factors and have not analyzed the entire genome. However, whole mammalian genome arrays have recently become commercially available from companies like Nimblegen. In the future, as ChIP-on-chip arrays become more and more advanced, high resolution whole genome maps of DNA-binding proteins and chromatin components for mammals will be analyzed in more detail.
== Alternatives == Introduced in 2007, ChIP sequencing (ChIP-seq) is a technology that uses chromatin immunoprecipitation to crosslink the proteins of interest to the DNA but then instead of using a micro-array, it uses the more accurate, higher throughput method of sequencing to localize interaction points. DamID is an alternative method that does not require antibodies. ChIP-exo uses exonuclease treatment to achieve up to single base pair resolution. CUT&RUN sequencing uses antibody recognition with targeted enzymatic cleavage to address some technical limitations of ChIP.
== References ==
== Further reading == Johnson, W. E.; Li, W.; Meyer, C. A.; Gottardo, R.; Carroll, J. S.; Brown, M.; Liu, X. S. (2006). "Model-based analysis of tiling-arrays for ChIP-chip". Proceedings of the National Academy of Sciences. 103 (33): 12457–12462. Bibcode:2006PNAS..10312457J. doi:10.1073/pnas.0601180103. ISSN 0027-8424. PMC 1567901. PMID 16895995. Benoukraf, Touati; Cauchy, Pierre; Fenouil, Romain; Jeanniard, Adrien; Koch, Frederic; Jaeger, Sébastien; Thieffry, Denis; Imbert, Jean; Andrau, Jean-Christophe; Spicuglia, Salvatore; Ferrier, Pierre (2009). "CoCAS: a ChIP-on-chip analysis suite". Bioinformatics. 25 (7): 954–955. doi:10.1093/bioinformatics/btp075. ISSN 1460-2059. PMC 2660873. PMID 19193731.
== External links == http://www.genome.gov/10005107 ENCODE project Chip-on-Chip (CoC) Package Information from Amkor Technology
=== Analysis and software === [1] CoCAS: a free Analysis software for Agilent ChIP-on-Chip experiments [2] rMAT: R implementation from MAT program to normalize and analyze tiling arrays and ChIP-chip data.