Native genomic blotting: high-resolution mapping of DNase I-hypersensitive sites and protein-DNA interactions.
DNase I-hypersensitive sites are observed in the promoter regions of actively expressed genes, potentially active genes, and genes that were once active. We have developed an approach that greatly increases the resolution for mapping these sites by electrophoresing genomic DNA on native polyacrylamide gels prior to electroblotting and hybridization. This improved method has been used to scan the promoter and coding region of a cell-cycle-dependent human histone H4 gene with an accuracy of +/-5-10 base pairs. Protein-DNA interactions can be seen in the autoradiograph as light areas and DNase I-hypersensitive sites as dark bands. Therefore, this method provides a rapid and relatively simple means to accurately localize protein-DNA interactions as well as DNase I-hypersensitive sites, thus directly displaying DNase I hypersensitivity and protein-DNA complexes on one autoradiograph. It also potentially allows the analysis of small changes in DNase I-hypersensitive sites under various biological conditions. With this technique rather large regions of DNA can be screened to determine areas that should be analyzed by more sophisticated methods, such as genomic sequencing or gel retardation assays.
University of Florida College of Medicine, Gainsville 32610.
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Pauli
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Chrysogelos
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Stein
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Stein
D004273:DNA, Neoplasm / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D005796:Genes; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D009710:Nucleotide Mapping; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013379:Substrate Specificity
D004273:DNA, Neoplasm; D003850:Deoxyribonuclease I