Four milliliter of saliva was collected from each participant in Oragene collection tubes (DNA Genotek, Kanata, Ontario). DNA was isolated following manufacturer's instructions. Briefly, each 4 ml saliva sample was pre-warmed overnight at 50°C. After incubation, each sample was divided into 4 × 1 ml aliquots in 1.5 ml tubes. Forty ul of manufacturer supplied isolation regent was added to each 1 ml aliquot and the tubes were briefly vortexed, incubated on ice for 5 min and centrifuged at 15,000 rpm for 5 min in a benchtop centrifuge. After centrifugation, the supernatant was transferred into a fresh 15 ml Falcon tube and 4 ml of 100% ethanol were added to each tube (less if the original saliva volume was less than 4 ml). Samples were mixed by inversion 15–20 times, incubated at room temperature for 15–30 min and centrifuged again at 15,000 rpm for 5 min. The supernatant was removed from each tube and the pellet was allowed to air dry at room temperature for at least 2 h. During the first year sampling, dried pellets were resuspended in 1
