SimpleChIP? Human H19/Igf2 Imprinting Control Region Primers

• 产品编号：CST-5172S      品牌：CST       原厂货号：5172S
• 产品分类：PCR/RT-PCR/qRT-PCR > 其它 > 引物 > 其他引物与寡核苷酸
• 应用分类：

描述：

SimpleChIP® Human H19/Igf2 Imprinting Control Region Primers包含有针对于人H19/Igf2印迹对照区（ICR）特异的正反向PCR引物。该引物可用于用染色质免疫沉淀分离出的DNA扩增。引物已经最优化，适合用于SYBR® Green实时定量PCR。并且已经与SimpleChIP® Enzymatic Chromatin IP Kits #9002和ChIP-验证抗体一起进行了测试。H19/Igf2基因被结合有CTCF的印迹对照区调控。

染色质免疫沉淀试验（ChIP）是一个在细胞自然状态下检测DNA和蛋白质相互作用的一个有效技术（1,2）。该试验可以用于分离出与基因组特定区域结合的一组蛋白质，也可以用于鉴定某个特定蛋白在基因组上的多个结合位点（3-6）。ChIP可以用于确定基因启动子上的蛋白招募顺序，也可以“测定”整个基因组上某个组蛋白修饰的相对含量（3，4）。除了组蛋白，ChIP还可以用于分析染色体上结合的转录因子，辅转录因子，DNA复制因子和DNA修复蛋白。在做ChIP实验的时候，首先用甲醛固定细胞，甲醛用于将可逆的DNA－蛋白质交连反应固定化，保持住细胞内正在发生的DNA－蛋白质结合反应（1,2）。然后对细胞进行裂解，收集染色质，并用超声波法或酶切法对染色质片段化。片段化的染色质用特定蛋白或特定组蛋白修饰的抗体进行免疫沉淀反应。与特定蛋白或特定组蛋白修饰的DNA片断在染色质交连过程中被富集下来。免疫沉淀后，DNA-蛋白质的结合被打开，并对沉淀下来的DNA进行纯化。对纯化的DNA进行常规PCR或RT-PCR来测定目标DNA序列的数量（1,2）。除此之外，ChIP还可以与全基因组微阵列技术(ChIP on chip)，高通量测序技术（ChIP-Seq）或克隆技术相结合，进行全基因组的DNA－蛋白质结合分析和组蛋白修饰分析（3－8）。SimpleChIP®的引物已经为扩增ChIP实验的DNA反应实现了最优化，并且产品中提供了阳性和阴性对照，来确认ChIP实验的成败。

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 H19/Igf2 Imprinting Control Region Primers contain a mix of forward and reverse PCR primers that are specific to the human H19/Igf2 imprinting control region (ICR). 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 H19/Igf2 genes are regulated by an imprinting control region that binds CTCF.

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.