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From Biolecture.org
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<p>The cellular processes of transcription, DNA replication and DNA repair involve the interaction between genomic DNA and nuclear proteins. It had been known that certain regions within chromatin were extremely susceptible to DNase I digestion, which cleaves DNA in a low sequence specificity manner. Such hypersensitive sites were thought to be transcriptionally active regions, as evidenced by their association with RNA polymerase and topoisomerase I and II. It is now known that sensitivity to DNAse I regions correspond to regions of chromatin with loose DNA-histone association. Hypersensitive sites most often represent promoters regions, which require for DNA to be accessible for DNA binding transcriptional machinery to function.</p>
<h4><strong>ChIP-Chip and ChIP-Seq</strong></h4> <p>Histone modification was first detected on a genome wide level through the coupling of chromatin immunoprecipitation (ChIP) technology with DNA microarrays, termed ChIP-Chip. However instead of isolating a DNA-binding transcription factor or enhancer protein through chromatin immunoprecipitation, the proteins of interest are the modified histones themselves. </p> <p>① Histones are cross-linked to DNA in vivo through light chemical treatment.</p> <p>② The cells are next lysed, allowing for the chromatin to be extracted and fragmented, either by sonication or treatment with a non-specific restriction enzyme.</p> <p>③ Modification-specific antibodies in turn, are used to immunoprecipitate the DNA-histone complexes. </p> <p>④ Following immunoprecipitation, the DNA is purified from the histones, amplified via PCR and labeled with a fluorescent tag.</p> <p>⑤ The final step involves hybridization of labeled DNA, both immunoprecipitated DNA and non-immunoprecipitated onto a microarray containing immobilized gDNA.</p> <p>⑥ Analysis of the relative signal intensity allows the sites of histone modification to be determined. </p>
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