Comparison of two DNA Extraction Protocols Representing Bacterial Community from Bulk Soil by PCR-DGGE

Abstract

Aims: The objective of this work was to investigate the efficiency of DNA extraction from bulk soil using the Britânia® mini mixer combined with phenol extraction to study bacterial communities, from three sampling sites localized in São Gonçalo-RJ, Brazil, comparing it to a commercial kit by the PCR-DGGE technique. Study Design: Molecular fingerprints of bacterial communities in bulk soil from three sampling sites were generated by DGGE after 16S rDNA gene amplification. Place and Duration of Study: Faculdade de Formação de Professores-UERJ and Embrapa Agrobiologia between April 2010 and April 2013. Methodology: Samples of DNA, in triplicate, extracted from bulk soil at three sampling sites localized in São Gonçalo-RJ, Brazil, were obtained by two protocols of DNA extraction. The DNA samples were used as template to amplify the 16S rDNA gene using specific primers to α and β-Proteobacteria groups. The PCR products were used for a second amplification using the primers F968GC and R1401 for subsequent DGGE analysis. The products from the second amplification were subjected to DGGE and band patterns were statistically analysed. Results: The band profiles obtained from the protocol that used a hand held mini mixer were intense and showed higher similarity among the triplicates from the sampling sites. Although both methods were capable of achieving a representative DNA profile from the soil bacterial community, the band patterns produced by them for the same areas were different. Conclusion: The DGGE profile of specific groups such as α- and β-Proteobacteria was a useful tool to compare the two soil DNA extraction protocols and also to compare the community structure of the different sampled areas. The DNA extraction protocol that used the Britânia® mini mixer produced band profiles with higher values of richness, but missed some bacterial targets as the commercial kit did. Both protocols have validity for the study of bacterial communities in bulk soil. The clusters of band profiles obtained via 16S rDNA PCR-DGGE indicated differences in bacterial communities of bulk soil from the three sampling sites for different ecological succession stages localized in São Gonçalo, RJ. The diversity analysis showed that the α-Proteobacteria group was predominant in bulk soil from these sites.