A rapid in vitro polyomavirus DNA replication assay.
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 (32)P-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.
Department of Tropical Medicine and Medical Microbiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Atherton Building, 3675 Kilauea Avenue, Honolulu, HI 96816, USA.
Katja
K
Ziegler
Thomas
T
Bui
Richard J
RJ
Frisque
Andrew
A
Grandinetti
Vivek R
VR
Nerurkar
D004279:DNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D011120:Polyomavirus / Q000235:genetics / Q000502:physiology*; D014773:Virology / Q000379:methods*; D014779:Virus Replication
D004279:DNA, Viral; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific