In Vivo Dimethyl Sulfate (DMS) Footprinting via Ligation-Mediated Polymerase Chain Reaction (LM-PCR)
Adapted from Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 2nd edition, by Michael F. Carey, Craig L. Peterson, and Stephen T. Smale. CSHL Press, Cold Spring Harbor, NY, USA, 2009.INTRODUCTION
Ligation-mediated polymerase chain reaction (LM-PCR) is a powerful technique for detecting DNA strand breaks within a complex sample because of its extreme sensitivity and specificity. This general method has proven to be invaluable for several purposes, in particular, in analysis of the properties of an endogenous DNA locus. LM-PCR can be coupled to a number of DNA modification and cleavage reagents. Coupling to dimethyl sulfate (DMS) modification, described here, results in assays that are analogous to in vitro DNase I footprinting or methylation protection. In this method, DMS is added directly to intact cells. After quenching the modification reaction, genomic DNA is purified, and cleavage adjacent to modified bases is induced by piperidine treatment. LM-PCR is then performed on the cleaved genomic DNA.
