TENT: A Rapid DNA Extraction Method of Staphylococcus aureus

Recently different methods are introduced for DNA extraction from bacteria. Bacterial DNA can be extracted using a commercial kit and conventional (e.g., boiling, phenol-chloroform and detergent methods). In the manual methods, chemically solution such as EDTA, SDS, NaCl, Tris hydrochloride, phenol-chloroform, proteinase K and lysozyme can be proposed (1). The phenol-chloroform DNA extraction method is time-consuming and manipulation of toxic solvents may be hazardous to the environment and the technician. Furthermore, several washing and centrifugation steps increases the risk of sample contamination (2). Therefore, several methods have been introduced as alternative techniques to the phenol-chloroform method including boiling and detergent methods. These approaches are convenient and low-cost, but due to remaining of some residual protein with the DNA, the purity of the extracted DNA is poor. Commercial DNA extraction kits offer a low risk of contamination and they are faster than conventional protocols, but the amount of DNA recovered is highly variable (3). We examined TENT (Tris-EDTANaCl-TritonX100) buffer for DNA extraction from Staphylococcus aureus and DNA purity compared to detergent and kit methods. In this study, S. aureus ATCC 29247 was cultured on tryptic soy agar (TSA) at 37 °C for 24 h and used in different DNA extraction methods. Pure colonies were suspended in 300 μL of TENT buffer (10 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, 5% [v/v] Triton X100, pH 8.0). The cell suspension was boiled at 100 °C and then centrifuged. Supernatant fluid was transferred into a new sterile tube. Subsequently, cold 95% ethanol was added to the supernatant and kept at −20 °C for 20 min. After this stage, the solution was centrifuged. DNA template was dissolved in 50 μl sterile distilled water and stored at −20 °C until PCR amplification. This step was repeated three times and the result were compared. Manual genomic DNA extraction of S. aureus was performed using detergent method (4). Genomic DNA extract by a kit, the product of the Viogene Company (UK) according to the manufacturer's instructions. Like before, this step was repeated three times. Purity of extracted DNA was deter-