Evaluation outcome of actinobacteria diversity in saline environment influenced by different DNA extraction methods

Abstract:

Abstract:［Objective］To evaluate the influence of DNA extraction methods on the actinobacteria diversity analysis in saline environment via 16S rDNA Restriction Fragment Length Polymorphism．［Methods］CTAB-SDS method，glass bead beating method and repeated freezing and thawing method were used to extract total DNA in soil samples from the Yanqi Salten． The 16S rDNA clone libraries were constructed by using the purified 16S rDNA PCR amplicons to transform the E．coli DH5α．The transformants in the library were further analyzed by RFLP．The unique 16S rDNA clones were sequenced and further used for phylogenetic analysis．［Results］Different Operational Taxonomic Units (OTU) were obtained from DNA extracts and total 35 OTUs were obtained from CTAB-SDS method，19 OTUs from galss bead beating method and 14 OTUs from repeated freezing and thawing methods．Up to 52% OTUs in the three libraries constructed displayed lower similarity with the published sequence，perhaps representing novel taxons．The total OTUs belong to Actinobacteridae，Acidimicrobidae and Rubrobacteridae subclasses．［Conclusion］ DNA extraction methods influence the actinobacterial diversity． Each of the DNA extraction method in our study has some drawbacks and biases，so it is better to use combined DNA extracts from different DNA methods to evaluate the microbial diversity in salty environments.

Key words:Keywords:saline environment，DNA extraction，16S rDNA，clone library，actinobacteria