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Quantitative Analysis of Protein-DNA Associations in Vivo Using Real-Time PCR, Rev A

Swarup K Chakrabarti and Raghavendra G Mirmira*, Departments of Medicine and Pharmacology, Division of Endocrinology and Metabolism, University of Virginia Health Sciences Center, 450 Ray C Hunt Dr, Box 801407, Charlottesville, VA 22903 USA

*Corresponding author


Introduction
The chromatin immunoprecipitation (ChIP) assay has been used in recent years to identify the association of histone complexes and transcription factors with specific genomic DNA targets in cells of Drosophila or yeast. As shown in Figure 1, the technique first involves the treatment of cells with formaldehyde or ultraviolet light to covalently crosslink proteins to DNA. The chromatin from the cells is then isolated and fragmented (by restriction digestion or sonication), and the protein of interest is immunoprecipitated using a specific antibody. The coimmunoprecipitated DNA fragments are then liberated from the protein by treatment with proteinase K, and the DNA fragments are identified by Southern blotting or PCR (Kuo and Allis 1999). More recently, studies have established the utility of the ChIP assay in studying histone complex associations and transcription factor binding in mammalian cells as well (Boyd and Farnham 1999). However, a potential barrier to the quantitative study of some mammalian transcription factors by the ChIP assay is their relatively low level of expression in cells, coupled with the rather large size of the mammalian genome compared to yeast or Drosophila. Southern blot detection of DNA does not offer the necessary sensitivity for such studies, nor is traditional PCR easily amenable to quantitation. However, we demonstrate here that real-time PCR wit
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