Genome-scale identification of active DNA replication origins.
Understanding the nature of DNA replication origins in metazoan is quite challenging. In the absence of a genetic assay like in yeast, methods were devised based on the DNA structure, the visualization or quantification of the first nascent strands that are synthesized at origins, or on the localization of origin binding proteins. The purification and quantification of RNA-primed nascent DNA at origins during initiation of DNA synthesis is the most exhaustive and precise method to map active replication origins at present. We have upgraded this method to the level of reproducibility and enrichment required for genome-wide analyses by microarrays or deep sequencing. We detail here the protocol and the controls required at the different steps.
Institute of Human Genetics, CNRS, Montpellier 34396, France.
Christelle
C
Cayrou
Damien
D
Grégoire
Philippe
P
Coulombe
Etienne
E
Danis
Marcel
M
Méchali
D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D053837:DNA Cleavage; D004261:DNA Replication; D005090:Exodeoxyribonucleases / Q000737:chemistry; D056426:Genetic Loci; D016678:Genome; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D059625:Liquid-Liquid Extraction; D020411:Oligonucleotide Array Sequence Analysis; D059367:RNA Cleavage; D060888:Real-Time Polymerase Chain Reaction; D018741:Replication Origin; D012259:Ribonuclease, Pancreatic / Q000737:chemistry
D018398:Homeodomain Proteins; D004247:DNA; D005090:Exodeoxyribonucleases; D012259:Ribonuclease, Pancreatic