Quantitative Comparison of Oral Site-specific DNA Isolates Reveals Differential Outcomes

Abstract

Introduction: More and more evidence has accumulated that suggests salivary sampling may provide direct analysis of oral conditions and microbial constituents, but may also be useful in the diagnosis and early detection of other chronic diseases. Although multiple methods of oral sampling currently exist, some methods are prohibitively expensive or based upon technologies not ubiquitously available at public health centers or state-funded colleges. This study provides a comparative analysis of DNA concentrations and quality from five specific oral sites derived using sterile paper points, including the gingival crevice between the upper central incisors, biofilm of the upper first molar, lingual incisor, and the dorsum of the tongue for comparison with unstimulated saliva collection. Methods: This study analyzed previously collected unstimulated saliva and paper point samples. In brief, DNA was isolated from each using TRIzol (phenol: Chloroform) extraction and DNA quantification and quality was measured using a NanoDrop spectrophotometer at 260 and 280 nm. Results: Analysis of Paper Point (PP) biofilm sampling sites from upper first molar, lower incisor, and dorsum of the tongue revealed similar average DNA concentrations, ranging between 14,342 ng/uL and 14,402 ng/uL (p=0.9851). Although variations were observed between different patients, samples from different oral sites within the same patient were strikingly similar, R=0.8355. Comparison of DNA isolated from fluids, gingival crevicular fluid (GCF) and unstimulated saliva revealed average DNA concentrations that were similar to the biofilm sampling sites (14,686 ng/uL and 13,743 ng/uL, respectively), which were not significantly different from one another (p=0.7893). DNA concentrations ranged considerably between patients (low = 4,410 ng/uL; high = 48,783 ng/uL), but were most similar with different samples (GCF, saliva) from the same patient (Pearson’s R=0.6979). In addition, DNA purity measured by A260:A280 nm absorbance did not reveal any significant difference among sampling sites (range 1.62 – 1.70; p=0.427). Discussion: Although many methods are available to provide oral sampling, simple and low-cost methods such as paper point sampling, unstimulated saliva collection and buccal swabs may represent tools that provide sufficient DNA quality and quantity for molecular screening. In addition, although heterogeneity between patient samples will always be present – samples from various oral sites within the same patient may provide roughly equivalent DNA samples for further screening and molecular analysis.