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Modified and reliable procedure for non-invasive sampling and extraction of mouse DNA

We previously published a procedure for easy and non-invasive sampling of DNA from laboratory mice in this journal (Meldgaard et al. 2004). Subsequently, we found that the procedure did not always ensure DNA extraction from sampled buccal cells. Therefore, it became necessary to modify the DNA extraction procedure slightly. The modified procedure, which is described here, reliably yields amplifiable genomic DNA. The original mouth swabbing procedure has not been changed; cotton sticks with 2 mm diameter buds (aluminium shaft 0.9 mm 150 mm; Applimed SA, Châtel-St-Denis, Switzerland) are used. A firm grip of the neck skin keeps the mouth of the mouse open and accessible to forceful inner cheek scraping. Following scraping of both inner cheeks, the cotton stick is cut close to the cotton bud and dropped down into a 1.5 mL microcentrifuge tube. While sampling, each tube is left open in a rack to allow the cotton bud to dry. For a negative control, a mock sample is taken, i.e. a cotton stick not applied for mouth swabbing is swept across a cage and subjected to DNA extraction along with the samples. We are now recommending the procedure for DNA extraction from the cotton buds as follows: To each tube 0.5 mL of phosphate-buffered saline (PBS) with 10 mmol/L ethylene diamine tetraacetic acid (EDTA) is aliquoted, and the cotton buds are soaked for 5 min, while the tubes are stirred a few times. Then the tubes are centrifuged briefly in a microcentrifuge. The application of PBS rather than water, as described in our original report, is assumed to prevent hypotonic lysis of epithelial cells sampled, and EDTA is known to chelate magnesium ions and thus prevent DNases from degrading DNA if released prematurely. Each tube is opened and the cotton bud is removed and disposed of while care is taken to leave behind as much liquid as possible by pressing the cotton bud against the interior of the microcentrifuge tube. The released epithelial cells are precipitated by centrifuging the tubes at 4000 g for 4 min. Check that a vague pellet is visible in each of the tubes at this point, except for the tube representing the mock sample. Supernatants are pipetted off while taking great care not to disturb the cell pellets. Then each pellet is resuspended in 28 mL of 0.1 mmol/L potassium hydroxide (freshly prepared) and incubated for 20 min at 758C on a heating block, while the tubes are covered with a piece of aluminium foil to reduce condensation on the inside of the lids. The extended incubation time was found to be absolutely essential for reliable DNA extraction. To neutralize and dilute extracts 10-fold, 252 mL of 20 mmol/L Tris–HCl (pH 7.5) is added to each tube at room temperature, and the extracts are used for polymerase chain reaction.