Two-dimensional gel electrophoretic method for mapping DNA replicons.
We describe in detail a method which allows determination of the directions of replication fork movement through segments of DNA for which cloned probes are available. The method uses two-dimensional neutral-alkaline agarose gel electrophoresis followed by hybridization with short probe sequences. The nascent strands of replicating molecules form an arc separated from parental and nonreplicating strands. The closer a probe is to its replication origin or to the origin-proximal end of its restriction fragment, the shorter the nascent strands that are detected by the probe. The use of multiple probes allows determination of directions of replication fork movement, as well as locations of origins and termini. In this study, we used simian virus 40 as a model to demonstrate the feasibility of the method, and we discuss its applicability to other systems.
Nawotka
K A
KA
Department of Molecular and Cellular Biology, Roswell Park Memorial Institute, Buffalo, New York 14263.
Huberman
J A
JA
D000818:Animals; D002460:Cell Line; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods; D009693:Nucleic Acid Hybridization; D012093:Replicon; D013539:Simian virus 40 / Q000235:genetics*
D004279:DNA, Viral; D004262:DNA Restriction Enzymes