title: Mapping origins of DNA replication in eukaryotes.
abstract: Methods are described here to map an origin of replication in eukaryotes. Replicating DNA is enriched by BND cellulose column chromatography and by lambda-exonuclease digestion; this approach has largely superceded enrichment by BrdU incorporation. The general area in which replication begins can be deciphered by neutral/neutral 2D gel electrophoresis: a restriction fragment containing the replication bubble will form a bubble arc on these gels. A more sensitive method employs PCR analysis of nascent strands that are size-fractionated. Once the general area containing the origin of bidirectional replication has been mapped, a finer level of resolution can be obtained by replication initiation point (RIP) mapping, in which start sites of DNA synthesis are identified at the nucleotide level.
authors.affiliation: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA.
authors.forename: Susan A
authors.initials: SA
authors.lastname: Gerbi
mesh_terms: D000818:Animals; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002874:Chromosome Mapping / Q000379:methods; D003636:DEAE-Cellulose / Q000031:analogs & derivatives; D004247:DNA / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004261:DNA Replication; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005057:Eukaryotic Cells; D005090:Exodeoxyribonucleases; D016133:Polymerase Chain Reaction / Q000379:methods; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D018741:Replication Origin; D014764:Viral Proteins
chemical_list: D014764:Viral Proteins; D004247:DNA; D003636:DEAE-Cellulose; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda