In Cellulo DNA Analysis: LMPCR Footprinting

Abstract

The in cellulo analysis of protein-DNA interactions and chromatin structure is very important to better understand the mechanisms involved in the regulation of gene expression. The nuclease-hypersensitive sites and sequences bound by transcription factors often correspond to genetic regulatory elements. Using the ligation-mediated polymerase chain reaction (LMPCR) technology, it is possible to precisely analyze these DNA sequences to demonstrate the existence of DNA-protein interactions or unusual DNA structures directly in living cells. Indeed, the ideal chromatin substrate is, of course, found inside intact cells. LMPCR, a genomic sequencing technique that map DNA single-strand breaks at the sequence level of resolution, is the method of choice for in cellulo footprinting and DNA structure studies because it can be used to investigate complex animal genomes, including human. The detailed conventional and automated LMPCR protocols are presented in this chapter.

Keywords: DNA polymerase; DNA-protein interaction; Deoxyribonuclease I; Dimethylsulfate; Footprints; Ligation-mediated polymerase chain reaction; Living cell; Polymerase chain reaction; Ultraviolet light C.