Mapping origins of DNA replication in eukaryotes.
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DNA is enriched by BND cellulose column chromatography and by lambda-exonuclease digestion; this approach has largely superceded enrichment by BrdU incorporation.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.
Gerbi
Susan A
SA
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA.
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
D014764:Viral Proteins; D004247:DNA; D003636:DEAE-Cellulose; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda