Optimization of Pre-Sampling Conditions for Salivary Genomic DNA Extraction: A Single-Donor Analysis

OBJECTIVES: Saliva-based diagnostics are expanding, yet the impact of common pre-sampling behaviors on saliva-derived genomic DNA (gDNA) recovery is not well quantified. This pilot study aimed to assess how tooth brushing, gargling, and water intake influence salivary gDNA yield and purity within a controlled single-donor setting. METHODS: Eight pre-sampling conditions were evaluated using all combinations of tooth brushing, gargling, and water intake. Saliva was collected in triplicate per condition, and cells were isolated for gDNA extraction using a Qiagen kit (Cat. No. 158023). Main effects and interactions on DNA yield and purity were analyzed by three-way ANOVA. RESULTS: Tooth brushing was the dominant contributor to variability in salivary DNA yield, accounting for 62.9% of the total variation, more than 11-fold greater than gargling. Water intake showed no significant effect on yield. Controlling brushing status reduced within-donor variability in DNA recovery across conditions. CONCLUSIONS: In this single-donor pilot analysis, tooth brushing status exerted the strongest influence on salivary gDNA yield, whereas water intake had minimal impact. These results provide quantitative guidance for controlling pre-sampling behaviors to improve within-sample consistency in saliva gDNA extraction and motivate follow-up validation in larger, multi-donor cohorts.