A rapid in vitro polyomavirus DNA replication assay

Katja Ziegler, Thomas Bui, Richard J. Frisque, Andrew Grandinetti, Vivek R. Nerurkar

Research output: Contribution to journalArticlepeer-review

57 Scopus citations


Traditionally, the Hirt extraction method, a multi-step, labor-intensive and time-consuming procedure, is employed to extract selectively low-molecular weight DNA for polyomavirus DNA replication analyses. DNA replication results obtained with this approach are often inconsistent between replicate samples. To increase the efficiency and reproducibility of the polyomavirus DNA replication assay, we compared the DNA quality and yield using Qiagen Spin Column technology and the Hirt extraction technique. CV-1 cells transfected with SV40 DNA were harvested at days 2, 4, and 6 post-transfection, and DNA was extracted using the Qiagen Spin Column and the Hirt extraction methods. Southern hybridization was performed using a 32P-labeled linear full-length SV40 DNA probe. Viral DNA replication was quantitated using a BioRad phosphorimager, and results obtained with the two procedures were compared. Southern blot analysis revealed consistent and enhanced SV40 DNA recovery using the Qiagen Spin Column technology, and viral DNA replication over a 6-day period was reproducible among triplicate samples. In addition, Qiagen Spin Column technology reduced the time required to obtain good quality DNA for polyomavirus replication assays from 24 h to less than 3 h. Adoption of this extraction procedure will improve the determination of polyomavirus DNA replication activity, while reducing the investigator's exposure to and disposal of toxic organic compounds.

Original languageEnglish (US)
Pages (from-to)123-127
Number of pages5
JournalJournal of Virological Methods
Issue number1
StatePublished - Dec 1 2004

All Science Journal Classification (ASJC) codes

  • Virology


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