Optimizing Bacterial DNA Isolation from Oral Samples

Molecular methods for studying complex microbial communities are continuously improving, but these techniques are dependent on obtaining a representative bacterial DNA sample. Bacterial DNA extraction methods may have biases including incomplete bacterial cell lysis and loss of DNA. The ability to freeze samples for later analysis is a major advantage, but there is evidence that freezing biological samples can result in significant loss of bacterial DNA. Objectives: Our purpose was to compare bacterial DNA yields obtained from different methods of DNA isolation and to compare DNA yields among fresh and frozen samples. Methods: Pooled supragingival plaque was collected and divided to provide replicates for each experimental protocol. Aliquots were processed using different bacterial DNA isolation protocols, including bead beating and commercial bacterial DNA isolation kits. Fresh samples were analyzed immediately and frozen samples were analyzed after thawing. DNA yields were measured using a Nanodrop® spectrophotometer. In addition DNA was run on a 1% agarose gel and visualized. Results: Bead beating consistently gave higher bacterial DNA yields than the commercial kits tested, but resulted in lower molecular weight DNA. 1% SDS was added to the sample before bead beating to inhibit nucleases, and resulted in high molecular weight DNA. Commercial kits provided consistent yields of high molecular weight DNA. Frozen samples that were processed after thawing by first centrifuging and discarding supernatants had lower DNA yields than frozen samples that were processed so that supernatants were retained and fresh samples. Conclusions: Bead beating provided the greatest DNA yield among the methods tested for bacterial DNA isolation from dental plaque, but it appears that bead beating released nucleases that cleaved DNA molecules. Freezing samples did not result in loss of DNA if the samples were processed so that supernatants were retained. Supported by NIH DE16125, DE10467, NRSA T32DE14320.