Development of a Quick DNA Extraction Method for Streptococcus mutans

Objectives: AP-PCR is a common method used to obtain DNA fingerprint profiles of S. mutans(SM) in epidemiology studies where large numbers of subjects are involved. Conventional DNA extraction methods involving commercial kits are time consuming and cost prohibitive in these large studies. The aim of this study was to develop and compare a new, quick DNA extraction method for SM. Methods: The assay involved extracting DNA from SM ATCC strain 25175 and 12 SM clinical isolates using the quick extraction method and a Masterpure DNA extraction kit. The quick extraction method utilizes EDTA to increase cell membrane permeability. In the quick extraction method the SM isolate is cultured, pelleted, and suspended in Tris-EDTA extraction buffer and incubated. Cell suspensions were mixed for 1 minute and then centrifuged at 12000 g for 10 minutes. Concentration and purity of the DNA was measured and AP-PCR was carried out for each of the DNA extracts using primer OPA 2. Results: Mean DNA concentration for the quick extraction was 773.4 ng/µl. Quick extractions resulted in an average 260/230 of 2.13 and an average 260/280 of 2.11. Masterpure extractions resulted in an average 260/230 of 2.13 and an average 260/280 of 2.02. AP-PCR fingerprints of the DNA extracts were analyzed for band matching as well as clonal type differentiation. Comparison of the different extracts from SM ATCC 25175 revealed identical band matching whereas band matching for the 12 clinical isolates showed a few variations. However, a clonal typing analysis of the fingerprints demonstrated the same clonal differentiation. Conclusions: The results suggest that the quick extract method may be a reliable and reproducible DNA extraction method for AP-PCR of SM. Additional work is underway comparing methods and larger numbers of SM isolates. Supported by NIH 1 RO1 DE017736-01A1.