A novel technique with enhanced detection and quantitation of HPV-16 E1- and E2-mediated DNA replication.
Transient DNA replication assays to detect papillomavirus E1/E2-mediated DNA replication have depended upon Southern blotting. This technique is hazardous (radioactive), labour intensive, semiquantitative, and physically limited in the number of samples that can be processed at any one time. We have overcome these problems by developing a real-time PCR protocol for the detection of E1/E2-mediated transient DNA replication. The results demonstrate detection of replication at levels not seen using Southern blotting demonstrating enhanced sensitivity. This technique is also, by definition, highly quantitative. Therefore, the real-time PCR technique is the optimal method for the detection of E1/E2-mediated DNA replication.
Taylor
Ewan R
ER
Institute of Comparative Medicine, Department of Veterinary Pathology, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow G61 1QH, Scotland.
Morgan
Iain M
IM
D045744:Cell Line, Tumor; D004261:DNA Replication; D004268:DNA-Binding Proteins; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase; D014162:Transfection
D004268:DNA-Binding Proteins; C093478:E1 protein, Human papillomavirus 16; C054855:E2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D019914:Taq Polymerase