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PASSINI, M. R. Z. The improvement of a technique for seawater DNA extracting and the comparison of DNA extraction methods for the characterization of a marine bacterial community. 2015. 91 f. Dissertação (Masters thesis Biotechnology) – Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2015. Marine microorganisms are the major pillars for life on Earth, since they are responsible for roles in biogeochemical cycles that no other organism can perform. Their metabolic products can be used in biotechnological applications and this is one of the main driving force to study them. With the limitations of culture-based techniques, it has become necessary to use molecular techniques to better characterize microbial genetic resources. However, these studies are done using directly environmental DNA, and therefore we can find different results depending on the methodology chosen for the DNA extraction. Thus, this study aimed to improve a technique for extracting DNA from sea water, in order to obtain a high quality DNA, high yield and a low time consuming protocol. Furthermore, to demonstrate the importance of the DNA extraction methods in the study of microorganisms by cultivation-independent methods, the diversity of a marine bacterial community was characterized by DGGE technique using different methods of DNA extraction. Initially, it was performed the improvement of different steps of the methodology of DNA extraction from water: sample processing, cell lysis, purification and recovery of DNA. Later, we compared five methods of DNA extraction for the characterization of a marine bacterial community by DGGE. The improved methodology of DNA extraction from seawater resulted in a protocol similar in DNA quality, with better yield and approximately four times faster than the protocol initially described. Regarding the characterization by different DNA extraction protocols, the analysis by DGGE resulted in distinct marine microbial community profiles, not only with respect to the presence or absence of DNA bands, but also to their intensity. However, was not observed significant differences in richness and diversity among the methodologies. Comparing the extraction methods, most of the OTUs were found in all methodologies, however, unique OTUs were found, demonstrating that the extraction techniques may cause bias toward the observation of some species.

Aperfeiçoamento de uma técnica para extrair DNA de água do mar e comparação de métodos de extração de DNA na caracterização de uma comunidade bacteriana marinha.