Mapping of polyomavirus DNA replicative intermediates by two-dimensional gel analysis using chemiluminescent detection.
Py DNA replicative intermediates (RIs) were mapped using a neutral/neutral two-dimensional (N/N 2D) technique by both chemiluminescent and radioactive detection. The nonradioactive method provides similar sensitivity to radioactive methods while possessing the advantages of stability of labeled probes, faster processing, multiple exposures, easy disposal and safety associated with nonradioactive detection. Application of this method to 2D gel analysis requires some technical modification to reduce background and save the substrate. The patterns of Py RIs with Afl II digestion suggest that bidirectional replication occurs during Py DNA synthesis in cell culture, but other unexpected structures of DNA replication are also seen.
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Department of Molecular Biology and Biochemistry, University of California at Irvine 92717, USA.
Villarreal
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D000818:Animals; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis* / Q000378:metabolism; D004076:Digoxigenin / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008163:Luminescent Measurements; D051379:Mice; D011120:Polyomavirus / Q000235:genetics*; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D004076:Digoxigenin