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.

Constraints imposed by supercoiling on in vitro amplification of polyomavirus DNA / L. Laghi, A.E. Randolph, A. Malesci, C.R. Boland. - In: JOURNAL OF GENERAL VIROLOGY. - ISSN 0022-1317. - 85:11(2004 Nov), pp. 3383-3388. [10.1099/vir.0.80039-0]

Constraints imposed by supercoiling on in vitro amplification of polyomavirus DNA

L. Laghi
;
A. Malesci
Penultimo
;
2004

Abstract

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.
JC virus-DNA; progressive mutifocal leukoencephalopathy; viral DNA; simian-virus-40; sequences; cancers; tumors; genome; helix; brain
Settore MED/09 - Medicina Interna
Settore MED/12 - Gastroenterologia
nov-2004
Article (author)
File in questo prodotto:
File Dimensione Formato  
3383.pdf

accesso riservato

Tipologia: Publisher's version/PDF
Dimensione 197.21 kB
Formato Adobe PDF
197.21 kB Adobe PDF   Visualizza/Apri   Richiedi una copia
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/22639
Citazioni
  • ???jsp.display-item.citation.pmc??? 2
  • Scopus 6
  • ???jsp.display-item.citation.isi??? 6
social impact