Analysis of microbial communities’ genetic diversity through denaturing gradient gel electrophoresis

Abstract

Hiral Borasiya , Maulin P Shah

Phylum- classes and specific PCR primers were assayed for the production clone libraries and denaturing gradient gel electrophoresis (DGGE) analysis of complex bacterial communities. Primers were designed to specifically amplify 16S rRNA gene fragments of Bacteroidetes phyla, Planctomycetes and Firmicutes, three classes phylum Proteobacteria on Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria, and cyanobacteria (including chloroplast 16S rRNA genes). Specificity of seven pairs of primers was tested by producing clone library of environmental DNA samples from mesotrophic and oligotrophic environment. Five of the seven pairs of primers specifically amplified target 16S rRNA gene sequence. One exception was a Betaproteobacteria-Firmicutes specific primers, which were quite successful with coastal water samples mesocosm. Phylogenetic analysis of sequences Gammaproteobacteria clone library showed that coastal sample gave several clones clustered within clades, which belongs to the oligotrophic marine Gammaproteobacteria (OMG) group, shows that this group is not confined to oligotrophic environment. Comparison Bacterial diversity DNA sample on the environment of coastal and open sea using two or three step process of nested PCRDGGE significant differences were found in bacterial community. Using primers specific group provides higher resolution Genetic fingerprinting approach than existing DGGE primers