39] Mapping DNA-protein interactions in cells and nuclei: Genomic sequencing by template purification

Abstract

Publisher Summary This chapter presents a procedure for analyzing DNA-protein interactions on genomic DNA using template purification and genomic sequencing. The principles of the purification method and genomic sequencing are discussed in the chapter. Precise protocols for the treatment of cells with dimethyl sulfate (DMS) or KMnO4, and the isolation of nuclei for DNase I footprinting are presented. A number of genomic sequencing procedures, principally aimed at determining at the nucleotide level DNA-protein interactions on genomic DNA, have been developed. Although these sequencing procedures depend on different approaches, they all are difficult to carry out. The principal limitation is that a sequencing signal must be generated from a single-copy sequence which, for man, or mouse, is present in a total of about 3 × 109 bp of genomic DNA. Finally, protocols for the purification of a specific sequence from whole genomic DNA followed by detection of a sequence ladder using primer extension are also described.