While there are many published methods for microbial DNA isolation from fecal samples, the experts at Second Genome Solutions recommended the use of bead-beating methods and MO BIO® PowerSoil or MOBIO PowerMag™ isolation kits for isolation of DNA for use in microbiome analysis. With the MO BIO PowerSoil and PowerMag isolation kits, the final eluted DNA is enriched for microbial DNA, making PCR more sensitive. Human or animal epithelial cells will lyse quickly under the heating and beating conditions used for microbial lysis, releasing the free DNA into the supernatant. The free DNA is subjected to prolonged mechanical breakage resulting in small fragment sizes that are washed out of the column during the binding step. The microbial DNA remains as high molecular weight fragments and is retained on the column.
Non-bead beating methods are not as efficient at recover gram-positive bacterial DNA and should be avoided. In our experience bead-beading methods, like those used in the PowerSoil and PowerMag kit, generally yield approximately 100 µl of 2 to 20 ng/µl of total genomic DNA from 0.1 g of stool. Starting with 0.1 g to 0.25 g of human feces is ideal for successful isolation. For dry mouse feces, our experts recommend working with less starting material, closer to 0.1 g, or diluting the samples 1:4 or 1:5 so that sufficient lysis solution can be absorbed. They suggest using the beads provided in the kit and found other separately purchased glass or silica bead products to be unnecessary.
DNA yields from human stool retain approximately 2% human DNA. In contrast, other types of samples, like vaginal swabs or biopsy, may contain up to 80% human DNA post-isolation.
Removing PCR Inhibitors
Stool is similar to soil in that it contains a large amount of PCR inhibitors. Further, the concentration of inhibitors will vary between individuals based on their diet. These inhibitors often bind to the silica membranes of spin columns and co-elute with DNA. Samples with this problem will often show a brown or yellow color after purification and low purities when measured with spectrophotometry. Again, because of these properties, our experts recommended using the MO BIO PowerSoil or PowerMag DNA Isolation kit because it incorporates a specialized Inhibitor Removal Technology (IRT) to remove humic acids, polyphenols, polysaccharides, heme, and dyes, resulting in high purity DNA and more accurate microbial profiles after amplification.