Constraints imposed by supercoiling on in vitro amplification of polyomavirus DNA.
Previous attempts to identify oncogenic polyomaviruses in human cancers have yielded conflicting results, even with the application of PCR technology. Here, it was considered whether the topological features of the polyomavirus genome interfere with efficient PCR amplification. Plasmid and SV40 DNAs were used as a model system for comparing the amplification efficiency of supercoiled, circular relaxed and linear templates. It was found that detection of circular templates required 10 times more molecules than detection of identical but linear templates. Supercoiling hindered the in vitro amplification of SV40 circles by a factor of 10, and erratic amplification of supercoiled SV40 occurred with subpicogram amounts of template. Accordingly, topoisomerase I treatment of DNA improved the PCR detection of supercoiled SV40, significantly decreasing the number of false-negative samples. Previously described, yet controversial, polyomavirus presence in human tissues should be reconsidered and topoisomerase I-sensitive polyomavirus amplification might help to detect polyomavirus genomes in mammalian tissues.
Laghi
Luigi
L
Gastroenterology Unit and Research Laboratory, Istituto Clinico Humanitas, via Manzoni 56, 20089 Rozzano (MI), Italy.
Randolph
Ann E
AE
Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
Malesci
Alberto
A
Department of Internal Medicine, University of Milan, Italy.|Gastroenterology Unit and Research Laboratory, Istituto Clinico Humanitas, via Manzoni 56, 20089 Rozzano (MI), Italy.
Boland
C Richard
CR
Division of Gastroenterology, Baylor University Medical Center, Dallas, USA.
D045744:Cell Line, Tumor; D004264:DNA Topoisomerases, Type I / Q000494:pharmacology; D004278:DNA, Superhelical / Q000096:biosynthesis*; D004279:DNA, Viral / Q000032:analysis / Q000096:biosynthesis*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011120:Polyomavirus / Q000187:drug effects / Q000235:genetics*; D027601:Polyomavirus Infections / Q000821:virology; D013539:Simian virus 40 / Q000187:drug effects / Q000235:genetics*; D013698:Templates, Genetic; D014412:Tumor Virus Infections / Q000821:virology
D004278:DNA, Superhelical; D004279:DNA, Viral; D004264:DNA Topoisomerases, Type I