Application of polymerase chain reaction-denaturing gradient gelelectrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting

We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns obtained with target DNA extracted from a soil by direct and indirect methods. For this purpose, two direct extraction methods, i.e. cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, i.e. cell extraction followed by DNA extraction, and combined RNA/DNA extraction from the bacterial cell fraction, were performed. Crude extracts were purified and amplified using universal bacterial primers. PCR products were then analysed by DGGE, and similarity between the profiles obtained was determined by unweighted pair group with mathematical averages clustering. The results showed clear profiles that presumably represented the dominant bacterial fractions in the samples.The profiles generated by all four methods were similar,indicating that the methods were of approximately equal efficiency in the extraction of target DNA representative of the soil bacterial community. However.........