title: 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.
authors.affiliation: Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada. Regen.Drouin@USherbrooke.ca.|Division of Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12th Avenue North, Sherbrooke, QC, Canada, J1H 5N4. Regen.Drouin@USherbrooke.ca.
authors.forename: Régen
authors.initials: R
authors.lastname: Drouin
authors.affiliation: Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bastien
authors.affiliation: Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Millau
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA, USA.
authors.forename: François
authors.initials: F
authors.lastname: Vigneault
authors.affiliation: Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Paradis
mesh_terms: D002843:Chromatin / Q000737:chemistry / Q000235:genetics*; D004247:DNA / Q000737:chemistry; D053904:DNA Breaks, Single-Stranded; D018983:DNA Footprinting / Q000379:methods*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D004247:DNA
keywords: DNA polymerase; DNA-protein interaction; Deoxyribonuclease I; Dimethylsulfate; Footprints; Ligation-mediated polymerase chain reaction; Living cell; Polymerase chain reaction; Ultraviolet light C