Impact of different polymerase chain reaction (PCR) strategies on denaturing gradient gel electrophoresis- based analysis of bacterial communities in soils/sediments from the Northern Jiangsu Oil Field, China

Abstract

The selection of polymerase chain reaction (PCR) strategies is vital to a successful assessment of bacterial communities in soils/sediments by 16S rRNA gene-based denaturing gradient gel electrophoresis (DGGE) analysis. To obtain reliable information of the bacteria communities in soils/sediments from the Northern Jiangsu Oil Field (NJOF), the impact of six PCR strategies on DGGE analysis has been investigated. The results showed that one-step PCR approach with primer set 341f⊥GC/534r (Strategies 1 and 2) was not suitable for 16S rRNA gene amplification of the bacterial communities in the NJOF soils/sediments before DGGE analysis due to its non-specific DNA amplification and low efficiency of 16S rRNA gene amplification. Strategy 6 (one nested PCR approach with primer sets 27f/907r and 341f⊥GC/534r with a purification procedure) could be the most accurate assessment of community diversities, but only be suitable to perform DGGE analysis for a few samples and not for high-throughput DGGE analysis because it was time-consuming; and Strategies 5 (one nested PCR approach with a dilution procedure) and 3 (one two-step PCR approach with primer sets 341f /534r and 341f⊥GC/534r) could provide similar information on the bacterial diversity of the NJOF soils/sediments without a purification procedure comparing with Strategy 6. Therefore, we prefer to recommend Strategies 5 and 3 for high-throughput DGGE analysis, and have successfully obtained the useful information of bacterial communities in different NJOF soils/sediments by Strategies 5. Key words: Polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) analysis, bacterial diversity, soils/sediments, Northern Jiangsu Oil Field (NJOF).