Cho Byung-Kwan, Knight Eric M, Palsson Bernhard Ø
Department of Bioengineering, University of California-San Diego, La Jolla, CA, USA.
Methods Mol Biol. 2008;439:131-45. doi: 10.1007/978-1-59745-188-8_9.
Interactions between cis-acting elements and proteins play a key role in transcriptional regulation of all known organisms. To better understand these interactions, researchers developed a method that couples chromatin immunoprecipitation with microarrays (also known as ChIP-chip), which is capable of providing a whole-genome map of protein-DNA interactions. This versatile and high-throughput strategy is initiated by formaldehyde-mediated cross-linking of DNA and proteins, followed by cell lysis, DNA fragmentation, and immunopurification. The immunoprecipitated DNA fragments are then purified from the proteins by reverse-cross-linking followed by amplification, labeling, and hybridization to a whole-genome tiling microarray against a reference sample. The enriched signals obtained from the microarray then are normalized by the reference sample and used to generate the whole-genome map of protein-DNA interactions. The protocol described here has been used for discovering the genomewide distribution of RNA polymerase and several transcription factors of Escherichia coli.
顺式作用元件与蛋白质之间的相互作用在所有已知生物体的转录调控中起着关键作用。为了更好地理解这些相互作用，研究人员开发了一种将染色质免疫沉淀与微阵列相结合的方法（也称为ChIP-chip），该方法能够提供蛋白质-DNA相互作用的全基因组图谱。这种通用且高通量的策略首先通过甲醛介导的DNA与蛋白质交联，随后进行细胞裂解、DNA片段化和免疫纯化。然后通过反向交联从蛋白质中纯化免疫沉淀的DNA片段，接着进行扩增、标记，并与针对参考样品的全基因组平铺微阵列杂交。从微阵列获得的富集信号随后通过参考样品进行归一化，并用于生成蛋白质-DNA相互作用的全基因组图谱。此处描述的方案已用于发现大肠杆菌RNA聚合酶和几种转录因子在全基因组范围内的分布。
