Technology - Location Analysis
The genome-wide location analysis method we have developed (Ren et al., 2000; Lee et al., 2002, Ren et al. 2003) allows protein-DNA interactions to be monitored across the genome. The method combines a modified ChromatinImmunoprecipitation (ChIP) procedure, which has been previously used to study in vivo protein-DNA interactions at one or a small number of specific DNA sites (Aparicio, 1999; Orlando et al., 2000), with DNA microarray analysis. Briefly, cells are fixed with formaldehyde, harvested by sonication, and DNA fragments that are crosslinked to a protein of interest are enriched by immunoprecipitation with a specific antibody. After reversal of the crosslinking, the enriched DNA is amplified by LM-PCR, and the Klenow-labeled with a fluorescent dye. A sample of DNA that has not been enriched by immunoprecipitation is subjected to the same treatment with a different fluorophore, and both IP-enriched and unenriched pools of labeled DNA are hybridized to a single DNA microarray containing 13,000 intergenic regions.
We designed the array to encompass the 13,000 best characterized promoter regions spanning the region from approximately 600 bp upstream to 250 bp downstream of the most-upstream start site for mRNAs selected from NCBI's RefSeq database in June 2001.
Details of how the microarray was designed and produced can be found here.
Binding sites were analyzed using a standard error model, a description of which is found here.