DNA Extraction and Viral Detection in Mice

Total DNA was extracted from the kidneys of mice A, C, D, E, and F with commercially available kits (UltraClean^® Tissue & Cells DNA Isolation kit (MO BIO Laboratories, Inc, USA); DNEasy Tissue and Blood (Qiagen) according to the manufacturers’ instructions. For total DNA extracts from mouse A, a PCR reaction mixture contained the following components with final concentration of 20 mmol/L Tris-HCl (pH 8.5 at 25 °C), 50 mmol/L KCl, 2 mmol/L MgCl₂, 200 µmol/L each dNTP (dATP, dCTP, dTTP and dGTP), 0.5 U of Taq polymerase (Fisher Biotec, Australia) and 0.2 µmol/L each primers specific for murine polyomavirus VP2 protein-coding region [7 (link)] in a final PCR reaction volume of 25 µL. The PCR was run on a Peltier thermal cycler (MJ Research, United States) with the following temperature program: an initial denaturing step at 94 °C for 5 min, then 40 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s. The PCR products were electrophoretically separated in 2% agarose E-gels (Invitrogen, Australia) and visualized with UV light [6 (link)].
A broad-spectrum papillomavirus PCR was performed on DNA extracts from mice C, D, E, and F, according to the method described in Forslund et al. (1999) [8 (link)]. Multiply primed rolling circle amplification was also performed on these same samples, following the method of Rector and co-authors (2004, 2005) [3 (link),9 (link)].