Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens

Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons.Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences.We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities.The microbial community colonizing the human gastrointestinal (GI) tract is diverse [1] and plays an important role in digestion, production of essential vitamins, as well as protecting the GI tract from pathogen colonization [2,3]. Dietary approaches such as the ingestion of non-digestible oligosaccharides (prebiotics) and fermented food products containing live culture (probiotics) have been speculated to confer health benefits by enhancing the growth of beneficial intestinal bacteria [4]. The influence of diet on intestinal microflora has been largely studied using conventional microbiological techniques. Many limitations are associated with these techniques, but a significant drawback comes from their reliance on the identification of appropriate growth nutrients and conditions. Estimates indicate that only 20 - 40% [5] of the total intestinal