Constraints imposed by supercoiling on in vitro amplification of polyomavirus DNA

Luigi Laghi, Ann E. Randolph, Alberto Malesci, C. Richard Boland

Research output: Contribution to journalArticlepeer-review

Abstract

Previous attempts to identify oncogenic polymaviruses 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.

Original languageEnglish
Pages (from-to)3383-3388
Number of pages6
JournalJournal of General Virology
Volume85
Issue number11
DOIs
Publication statusPublished - Nov 2004

ASJC Scopus subject areas

  • Virology
  • Immunology

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