Abstract
BackgroundThe method of chromatin immunoprecipitation combined with microarrays (ChIP-Chip) is a powerful tool for genome-wide analysis of protein binding. However, a high background signal is a common phenomenon.ResultsReinvestigation of the chromatin immunoprecipitation procedure led us to discover four causes of high background: i) non-unique sequences, ii) incomplete reversion of crosslinks, iii) retention of protein in spin-columns and iv) insufficient RNase treatment. The chromatin immunoprecipitation method was modified and applied to analyze genome-wide binding of SeqA and σ32 in Escherichia coli.ConclusionsFalse positive findings originating from these shortcomings of the method could explain surprising and contradictory findings in published ChIP-Chip studies. We present a modified chromatin immunoprecipitation method greatly reducing the background signal.
Highlights
The method of chromatin immunoprecipitation combined with microarrays (ChIP-Chip) is a powerful tool for genome-wide analysis of protein binding
The results show that the same regions that gave a high background signal in the SeqA ChIP-Chip yielded a reduced signal if the DNA is crosslinked and reversed (Fig. 5C; compare blue and red signal, Fig. 3D-E)
Reinvestigation of σ32 binding to the E. coli genome Given the enormous background signal produced by the original ChIP-Chip method initially used in this study we considered it likely that published results based on this method would contain many false positives
Summary
The method of chromatin immunoprecipitation combined with microarrays (ChIP-Chip) is a powerful tool for genome-wide analysis of protein binding. Chromatin immunoprecipitation coupled with microarray analysis (ChIP-Chip) has become a widely used method for genome-wide localization of protein-DNA interactions [1]. The first step in the ChIP-Chip procedure is to fix protein-DNA interactions in living cells by chemical crosslinking (Fig. 1). After cell lysis the DNA is fragmented by sonication. This extract is subjected to immunoprecipitation (IP) with a specific antibody against the protein of interest. Two different fluorescence labels are used to label the IP DNA and a hybridization control DNA, respectively. Total DNA before IP (input DNA) is used as hybridization control.
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