1. Detecting episomal or integrated human papillomavirus 16 DNA using an exonuclease V-qPCR-based assay
- Author
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Julia E. Myers, Jason M. Bodily, Rona S. Scott, Katarzyna Zwolinska, Malgorzata Bienkowska-Haba, G. Raikhy, Martin Sapp, Joseph T. Guidry, K. Prasai, and Matthew L. Scott
- Subjects
Exodeoxyribonuclease V ,Virus Integration ,Biology ,Real-Time Polymerase Chain Reaction ,Genome ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Proviruses ,Virology ,Humans ,Human papillomavirus ,Cells, Cultured ,030304 developmental biology ,Human papillomavirus 16 ,0303 health sciences ,030302 biochemistry & molecular biology ,virus diseases ,Molecular biology ,female genital diseases and pregnancy complications ,genomic DNA ,Real-time polymerase chain reaction ,chemistry ,Exonuclease V ,Cell culture ,DNA, Viral ,DNA ,Plasmids - Abstract
Screening for human papillomavirus (HPV) integration into host cell chromosomes typically requires large amounts of time and reagents. We developed a rapid and sensitive assay based on exonuclease V (ExoV) and quantitative polymerase chain reaction (qPCR) to determine HPV genome configurations in cell lines and tissues. We established the assay using genomic DNA from cell lines known to harbor integrated or episomal HPV16. DNA was incubated with ExoV, which is specific for linear DNA, and the DNA fraction resistant to digestion was measured by qPCR. The percent of DNA resistant to ExoV digestion was calculated relative to undigested DNA for determination of episomal or integrated HPV16. The ExoV assay was accurate, capable of distinguishing episomal from integrated HPV16 in cell lines and tissues. Future applications of the ExoV assay may include screening of HPV genome configurations in the progression of HPV-associated cancers.
- Published
- 2019