An improved procedure for measuring DNA replication with transient assays in eukaryotic cells.
We describe an improved method for quantifying covalently closed circular DNAs replicated in eukaryotic cells. Normally, for such assays, plasmid DNAs transfected into animal cells must be extensively digested with restriction enzymes such as Dpn1 and the products of digestion of the input DNA separated from the Dpn1-resistant (replicated) DNAs by electrophoresis. In the procedure described, the input, unreplicated DNA is nicked by light digestion with Dpn1 and subsequently denatured and eliminated by S1 nuclease digestion. The replicated Dpn1-resistant DNA is detected by slot blot hybridization. Multiple samples can be rapidly assayed, with a considerable reduction in expense and labor.
Sullivan
M
M
Department of Microbiology, University of Texas, Austin 78712.
Folk
W R
WR
D000818:Animals; D002478:Cells, Cultured; D004261:DNA Replication; D004279:DNA, Viral / Q000032:analysis; D015151:Immunoblotting; D051379:Mice; D011120:Polyomavirus / Q000235:genetics; D014162:Transfection
D004279:DNA, Viral