Genome-wide mapping of the binding sites of proteins that interact with DNA.
Genome-wide mapping of the binding sites of proteins that interact with DNA.
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与 DNA 相互作用的蛋白质结合位点的全基因组图谱。
作者：
S. Spiro
通过特定转录因子协调调节一组基因的表达通常是细菌对环境信号做出反应或通过发育程序进行进展的能力的核心。因此，在许多情况下，鉴定在特定调节蛋白控制下的所有基因是有意义的。本章首先简要概述了用于尝试识别调节子的部分或全部成员的一些方法（即，作为转录激活因子或抑制因子的靶的那些基因）。此后，本章将集中在一种技术，染色质免疫沉淀和微阵列分析（ChIP芯片）和它的一些变种。提供了ChIP芯片实验的设计考虑和一些协议，沿着与下游数据分析相关的一些考虑。ChIP芯片是一种全基因组定位蛋白质结合位点的方法。在典型的ChIP芯片方案中，蛋白质在体内非特异性地与DNA交联。提取染色质并剪切，用合适的抗体免疫沉淀特异性蛋白质-DNA复合物。纯化后，将DNA与差异标记的参考样品一起杂交到微阵列（在一些方案中在扩增步骤之后）。微阵列上显示荧光比率升高的特征揭示了通过免疫沉淀富集的DNA序列。相应的基因组位置是富集的那些，因此接近结合位点。使用高密度平铺微阵列允许以相当高的分辨率进行结合位点定位。ChIP-chip很可能很快就会被ChIP-seq取代，在ChIP-seq中，免疫沉淀的DNA直接通过下一代测序技术进行分析。ChIP-chip和ChIP-seq的应用并不局限于调节蛋白，因为它们可以与任何直接或间接通过与另一种蛋白质的相互作用与DNA结合的蛋白质一起使用。因此，ChIP芯片已成功地用于定位类核蛋白和参与DNA复制的蛋白质的结合位点。
The coordinated regulation of the expression of a group of genes by a specific transcription factor frequently lies at the heart of the ability of a bacterium to respond to an environmental signal, or to progress through a developmental program. Thus, in many situations, it is of interest to identify all of the genes that are under the control of a particular regulatory protein. This chapter begins with a brief overview of some of the methods that have been used in attempts to identify some or all of the members of a regulon (i.e., those genes that are the targets for a transcriptional activator or repressor). Thereafter, the chapter will focus on one technique, chromatin immunoprecipitation and microarray analysis (ChIP-chip) and some of its variants. Design considerations and some protocols for ChIP-chip experiments are provided, along with some considerations related to downstream data analysis. ChIP-chip is a method for the genome-wide localization of protein-binding sites. In a typical ChIP-chip protocol, proteins are cross-linked nonspecifically to DNA in vivo. Chromatin is extracted and sheared, and specific protein-DNA complexes are immunoprecipitated with a suitable antibody. After purification, the DNA is hybridized to a microarray (after an amplification step in some protocols), together with a differentially labeled reference sample. Features on the microarray that show an elevated fluorescence ratio reveal DNA sequences that were enriched by immunoprecipitation. The corresponding genomic locations are those that were enriched, and are therefore close to sites of binding. The use of high-density tiled microarrays allows for binding site localization with quite high resolution. It is likely that ChIP-chip will soon be superseded by ChIP-seq, in which the immunoprecipitated DNA is analyzed directly by next-generation sequencing technologies. ChIP-chip and ChIP-seq applications are not confined to regulatory proteins, since they can be used with any protein that binds to DNA, either directly, or indirectly via an interaction with another protein. Thus, ChIP-chip has been used successfully to map binding sites for nucleoid proteins, and proteins involved in DNA replication.
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影响因子：
5.6
作者：
Cao, M;Kobel, PA;Helmann, JD
通讯作者：
Helmann, JD
ROMA：一种定义转录调节因子靶基因的体外方法。
DOI：
10.1016/j.ymeth.2008.10.009
发表时间：
2009
期刊：
Methods (San Diego, Calif.)
影响因子：
--
作者：
Maclellan,ShawnR;Eiamphungporn,Warawan;Helmann,JohnD
通讯作者：
Helmann,JohnD
影响因子：
10.5
作者：
Nonaka, Gen;Blankschien, Matthew;Rhodius, Virgil A.
通讯作者：
Rhodius, Virgil A.
影响因子：
16
作者：
Vora T;Hottes AK;Tavazoie S
通讯作者：
Tavazoie S