SimpleChIP? Human tRNA-Leu Anti-Codon (TAG) Primers

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• 产品分类：PCR/RT-PCR/qRT-PCR > 其它 > 引物 > 其他引物与寡核苷酸
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描述：

SimpleChIP® Human tRNA-Leu Anti-Codon (TAG) Primers中包含人tRNA-Leucine编码基因的特异性PCR引物。该引物可以用来扩增用ChIP分离到的DNA。该引物是经过优化的，可以用于SYBR® Green RT-PCR，这一点已在Cell Signaling Technology®用SimpleChIP® Enzymatic Chromatin IP Kits #9002 和#9003及ChIP抗体所验证。tRNA-Leu Anti-Codon (TAG)是POLR3A介导的转录的公认靶基因。ChIP是检测细胞内染色质中蛋白-DNA相互作用的通用方法(1,2)。该方法既可以用于检测基因组特定区域结合的多种蛋白，也可以检测与特定蛋白结合的多个基因组区域(3-6)。ChIP可以用来确认与某基因启动子区结合的多种蛋白的连接顺序，也可以测定整个基因座上特定组蛋白修饰的数量。除了组蛋白，ChIP还可以用于检测转录因子及其协同因子的结合，DNA复制子及DNA修复蛋白。在ChIP实验中，细胞先用多聚甲醛固定，多聚甲醛是一种能够维持细胞中蛋白-DNA交联原状的试剂，而且这种固定过程是可逆的(1,2)。细胞裂解后用用超声或酶切的方法收集染色质片段。片段化的染色质用特定蛋白的特异性抗体或组蛋白修饰抗体来进行免疫沉淀。与蛋白或修饰组蛋白结合的所有DNA片段都会作为染色质复合物被共沉淀捕获，而DNA序列的相对数量会在免疫选择过程中被富集和检测到。沉淀下来的蛋白-DNA交联复合物进行复性，就可以纯化得到DNA。沉淀下来的蛋白特异性的DNA常用标准PCR或qRT-PCR的方法来定量(1,2)。另外，ChIP也可以与genomic tiling micro-array (ChIP on chip)技术、高通量测序(ChIP-Seq)或克隆技术结合使用，以上这些技术都可以用于检测全基因组上的蛋白-DNA相互作用及组蛋白修饰(5-8)。SimpleChIP® primers适用于qPCR方法扩增ChIP相关的DNA，并且提供重要的阳性对照和阴性对照，确保ChIP实验的成功。

1. Orlando, V. (2000) Trends Biochem Sci 25, 99-104.
2. Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33.
3. Agalioti, T. et al. (2000) Cell 103, 667-78.
4. Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4.
5. Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.
6. Lee, T.I. et al. (2006) Cell 125, 301-13.
7. Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47.
8. Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.

原厂资料：

Description

SimpleChIP® Human tRNA-Leu Anti-Codon (TAG) Primers contain a mix of forward and reverse PCR primers that are specific to the human gene that encodes for tRNA-Leucine. These primers can be used to amplify DNA that has been isolated using chromatin immunoprecipitation (ChIP). Primers have been optimized for use in SYBR® Green quantitative real-time PCR and have been tested in conjunction with SimpleChIP® Enzymatic Chromatin IP Kits #9002 and #9003 and ChIP-validated antibodies from Cell Signaling Technology®. The tRNA-Leu Anti-Codon (TAG) gene is a well established target of POLR3A-mediated transcription.

Background

The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to either identify multiple proteins associated with a specific region of the genome or to identify the many regions of the genome bound by a particular protein (3-6). ChIP can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors, and DNA repair proteins. When performing the ChIP assay, cells are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. Fragmented chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or quantitative real-time PCR are often used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing (ChIP-Seq), or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8). SimpleChIP® primers have been optimized for amplification of ChIP-isolated DNA using real-time quantitative PCR and provide important positive and negative controls that can be used to confirm a successful ChIP experiment.

1. Orlando, V. (2000) Trends Biochem Sci 25, 99-104.
2. Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33.
3. Agalioti, T. et al. (2000) Cell 103, 667-78.
4. Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4.
5. Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.
6. Lee, T.I. et al. (2006) Cell 125, 301-13.
7. Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47.
8. Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.

注意事项：

Storage: Supplied in nuclease-free water at a concentration of
5 μM (each primer is at a final concentration of 5 μM). Store at
-20°C.