In cellulo DNA analysis (LMPCR footprinting).
The in cellulo analysis of DNA protein 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 any complex genomes, including human. The detailed conventional and automated LMPCR protocols are presented in this chapter.
Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, QC, Canada J1H 5N4.
Régen
R
Drouin
Nathalie
N
Bastien
Jean-François
JF
Millau
François
F
Vigneault
Isabelle
I
Paradis
D001483:Base Sequence; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D053904:DNA Breaks, Single-Stranded / Q000187:drug effects / Q000528:radiation effects; D018983:DNA Footprinting / Q000379:methods*; D019175:DNA Methylation / Q000187:drug effects / Q000528:radiation effects; D003850:Deoxyribonuclease I / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011740:Pyrimidine Dimers; D017422:Sequence Analysis, DNA; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D013698:Templates, Genetic; D014466:Ultraviolet Rays
D002843:Chromatin; D011740:Pyrimidine Dimers; D013463:Sulfuric Acid Esters; D004247:DNA; D003850:Deoxyribonuclease I; C007482:dimethyl sulfate