TY - JOUR
T1 - A rapid in vitro polyomavirus DNA replication assay
AU - Ziegler, Katja
AU - Bui, Thomas
AU - Frisque, Richard J.
AU - Grandinetti, Andrew
AU - Nerurkar, Vivek R.
N1 - Funding Information:
We thank Dr. Richard Yanagihara for critical comments and review of the manuscript. This work was supported by U.S. Public Health Service grants from the National Institute of Neurological Disorders and Stroke (S11 NS41833), and the Research Centers in Minority Institutions Program, National Center for Research Resources (G12 RR003061), National Institutes of Health.
PY - 2004/12/1
Y1 - 2004/12/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=5644237786&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=5644237786&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2004.08.012
DO - 10.1016/j.jviromet.2004.08.012
M3 - Article
C2 - 15488630
AN - SCOPUS:5644237786
SN - 0166-0934
VL - 122
SP - 123
EP - 127
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 1
ER -