|
ChIP-on-chip (also known as ChIP-chip) is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray (''"chip"''). Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA ''in vivo''. Specifically, it allows the identification of the cistrome, sum of binding sites, for DNA-binding proteins on a genome-wide basis. Whole-genome analysis can be performed to determine the locations of binding sites for almost any protein of interest.〔 As the name of the technique suggests, such proteins are generally those operating in the context of chromatin. The most prominent representatives of this class are transcription factors, replication-related proteins, like Origin Recognition Complex Protein(ORC), histones, their variants, and histone modifications. The goal of ChIP-on-chip is to locate protein binding sites that may help identify functional elements in the genome. For example, in the case of a transcription factor as a protein of interest, one can determine its transcription factor binding sites throughout the genome. Other proteins allow the identification of promoter regions, enhancers, repressors and silencing elements, insulators, boundary elements, and sequences that control DNA replication.〔M.J. Buck, J.D. Lieb, ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments, Genomics 83 (2004) 349-360.〕 If histones are subject of interest, it is believed that the distribution of modifications and their localizations may offer new insights into the mechanisms of regulation. One of the long-term goals ChIP-on-chip was designed for is to establish a catalogue of (selected) organisms that lists all protein-DNA interactions under various physiological conditions. This knowledge would ultimately help in the understanding of the machinery behind gene regulation, cell proliferation, and disease progression. Hence, ChIP-on-chip offers not only huge potential to complement our knowledge about the orchestration of the genome on the nucleotide level, but also on higher levels of information and regulation as it is propagated by research on epigenetics. == Technological platforms == The technical platforms to conduct ChIP-on-chip experiments are DNA microarrays, or ''"chips"''. They can be classified and distinguished according to various characteristics: * ''Probe type'': DNA arrays can comprise either mechanically spotted cDNAs or PCR-products, mechanically spotted oligonucleotides, or oligonucleotides that are synthesized ''in situ''. The early versions of microarrays were designed to detect RNAs from expressed genomic regions (open reading frames aka ORFs). Although such arrays are perfectly suited to study gene expression profiles, they have limited importance in ChIP experiments since most "interesting" proteins with respect to this technique bind in intergenic regions. Nowadays, even custom-made arrays can be designed and fine-tuned to match the requirements of an experiment. Also, any sequence of nucleotides can be synthesized to cover genic as well as intergenic regions. * ''Probe size'': Early version of cDNA arrays had a probe length of about 200bp. Latest array versions use oligos as short as 70- (Microarrays, Inc.) to 25-mers (Affymetrix). (Feb 2007) * ''Probe composition'': There are tiled and non-tiled DNA arrays. Non-tiled arrays use probes selected according to non-spatial criteria, i.e., the DNA sequences used as probes have no fixed distances in the genome. Tiled arrays, however, select a genomic region (or even a whole genome) and divide it into equal chunks. Such a region is called tiled path. The average distance between each pair of neighboring chunks (measured from the center of each chunk) gives the resolution of the tiled path. A path can be overlapping, end-to-end or spaced.〔Royce TE, Rozowsky JS, Bertone P, Samanta M, Stolc V, Weissman S, Snyder M, Gerstein M. Related Articles. Issues in the analysis of oligonucleotide tiling microarrays for transcript mapping. Trends Genet. 2005 Aug;21(8):466-75. Review.〕 * ''Array size'': The first microarrays used for ChIP-on-Chip contained about 13,000 spotted DNA segments representing all ORFs and intergenic regions from the yeast genome.〔 Nowadays, Affymetrix offers whole-genome tiled yeast arrays with a resolution of 5bp (all in all 3.2 million probes). Tiled arrays for the human genome become more and more powerful, too. Just to name example, Affymetrix offers a set of seven arrays with about 90 million probes, spanning the complete non-repetitive part of the human genome with about 35bp spacing. (Feb 2007) Besides the actual microarray, other hard- and software equipment is necessary to run ChIP-on-chip experiments. It is generally the case that one company’s microarrays can not be analyzed by another company’s processing hardware. Hence, buying an array requires also buying the associated workflow equipment. The most important elements are, among others, hybridization ovens, chip scanners, and software packages for subsequent numerical analysis of the raw data. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「ChIP-on-chip」の詳細全文を読む スポンサード リンク
|