Optimization of PCR‐DGGE technique to compare variations in human intestinal microflora community

Abstract

Advances in molecular techniques provide an advantage over conventional microbiological methods to study the diversity of human intestinal microflora. Variability in endogenous and dietary components makes extraction of bacterial DNA from feces a challenging task. The purity of DNA extracted from such a heterogeneous sample is crucial since it affects downstream analyses. In addition, both PCR and Denaturing Gradient Gel Electrophoresis (DGGE) methods must be optimized to perform meaningful comparative analyses. In this study, the relative efficacy of extracting bacterial DNA from feces was evaluated using: Mobio® DNA Stool Kit, QIAamp® DNA Mini Stool Kit, FastDNA® SPIN Kit for Soil, and FastDNA® SPIN Kit. These kits were found to yield 1.0 x 102, 1.1 x 102, 2.5 x 102, and 1.9 x 102 mg DNA per g feces, respectively. Optimum extraction yield was obtained using 10 to 25 mg of fecal sample (wet weight). Regardless of method, 16S rRNA gene of all samples was successfully amplified. Intestinal microflora community of four human subjects was evaluated using DGGE separation of the PCR products. Bacterial banding profiles showed similarity within each subject but high subject-to-subject variations. Bacterial DNA extracted using the Soil Kit produced the best profile in terms of clarity and number of bands. We conclude that using optimized conditions variations in human intestinal microflora community from fecal samples could be effectively compared using PCR-DGGE technique. Funded by Solae, LLC