Comparing DNA Extraction Methods for Human Microbiome

Six DNA extraction methods (Table 1) were compared in this study, representing different kinds and combinations of cell lysis mechanisms and DNA purification methods commonly used in the published literature on the human microbiome. Each method was evaluated using all 11 type strains and a mock community sample. The isolated genomic DNA was in a final volume of 200 µl.
Method 1. The QIAamp DNA mini kit (Qiagen, Valencia, CA) was used in this method with minor modifications. Briefly, 6 µl mutanolysin (25 KU/ml, Sigma-Aldrich) was added to a 500 µl aliquot of cells and the mixture was incubated for 30 min at 37°C. After this, 50 µl Proteinase K (20 mg/ml) and 500 µl AL buffer (Qiagen, Valencia, CA) were added and the sample was incubated for 30 min at 56°C. Then, 500 µl of ethanol was added and DNA was purified by using the columns provided in the kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.
Method 2. A two-step cell lysis procedure was employed before use of the QIAamp DNA mini kit (Qiagen, Valencia, CA). First, 50 µl lysozyme (10 mg/ml, Sigma-Aldrich), 6 µl mutanolysin (25 KU/ml, Sigma-Aldrich), and 3 µl lysostaphin (4000 U/ml, Sigma-Aldrich) were added to a 500 µl aliquot of cell suspension followed by incubation for 1 hour at 37°C. Second, 600 mg of 0.1-mm-diameter zirconia/silica beads (BioSpec, Bartlesville, OK) were added to the lysate and the microbial cells were mechanically disrupted using Mini-BeadBeater-96 (BioSpec, Bartlesville, OK) at 2100 rpm for 1 minute. Further isolation and purification of the total genomic DNA from lysates was done using QIAamp DNA mini kits (Qiagen, Valencia, CA).
Method 3. Genomic DNA was extracted by using the QIAamp DNA stool kit (Qiagen, Valencia, CA) with a 95°C lysis step according to the manufacturer's instructions. Briefly, 500 µl ASL buffer was add to a 500 µl aliquot of cells suspension and the mixture was heated for 5 min at 95°C. Then, 100 µl Proteinase K (20 mg/ml) and 1 ml AL buffer were added and the mixture was incubated for 10 min at 70°C. After this, 1 ml of ethanol was added and the rest of the isolation protocol was continued as described by the manufacturer.
Method 4. A 210 µl aliquot of 20% SDS, 500 µl of a mixture of phenol∶ chloroform∶ isoamyl alcohol (25∶24∶1)], and 600 mg of 0.1-mm-diameter zirconia/silica beads (BioSpec, Bartlesville, OK) were add to a 500 µl aliquot of cells suspension. Microbial cells were then disrupted by using Mini-BeadBeater-96 (BioSpec, Bartlesville, OK) set on 2100 rpm for 1 min. Next, the mixture was centrifuged at full speed (14000 rpm) for 5 min to separate phases. The top aqueous layer was transferred to a clean 2 ml micro-centrifuge tube. Then, 0.1 volume of 3 M sodium acetate and an equal volume of ice-cold isopropanol were added to the mixture. After incubation at −20°C for 10 min, the mixture was centrifuged at 4°C at 14,000 rpm for 15 min to collect the DNA pellet, which was then washed with 1 ml ice-cold 70% (v/v) ethanol and air dried. Finally, DNA pellets were re-suspended in 200 µl AE buffer (Qiagen, Valencia, CA).
Method 5. DNA was extracted by using the DNeasy Tissue Kit (Qiagen, Valencia, CA) and the manufacturer's protocol for isolation of genomic DNA from Gram-positive bacteria was followed. Briefly, 50 µl lysozyme (10 mg/ml, Sigma-Aldrich) was added to a 500 µl aliquot of cells and the mixture was incubated for 30 min at 37°C. After the addition of 50 µl Proteinase K (20 mg/ml) and 500 µl AL buffer, the mixture was incubated for 30 min at 56°C. Then, 500 µl of ethanol was added to the lysate and the genomic DNA was purified using the columns in the kit according to the manufacturer's instructions.
Method 6. In this method, an enzymatic lysis was conducted before the PowerSoil™ DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA) was used. Briefly, 50 µl of lysozyme (10 mg/ml, Sigma-Aldrich) was added to a 500 µl aliquot of bacterial cells followed by incubation for 1 hour at 37°C. The remainder of the DNA extraction was continued beginning with step 2 of the manufacturer's protocol.
This DNA extraction experiment was finished in 12 days, in which only one DNA extraction method was used per day. The selection of DNA extraction methods was made by randomly assigning each of the six DNA extraction methods to two of 12 days. On a given day, two experimenters used a given method to extract DNA from two replicates of each sample. This was repeated once, so eight replicate samples were analyzed using each method.