Application of nested PCR-DGGE (denaturing gradient gel electrophoresis) for the analysis of ciliate communities in soils.

ABSTRACT: Ciliates play important roles as prey and predators in ecosystems. Changes in the ciliate community can affect the composition and population of microfauna and microflora in ecosystems. To investigate the structure of ciliate communities, we developed a nested PCR-DGGE method, which combines a universal eukaryotic-specific primer set in the first PCR step with a ciliate-specific primer set in the second PCR step, to amplify 18S rRNA genes from ciliates. The 300 bp DGGE fragments generated more bands on the gel than the 600 bp DGGE fragments. Prior to bead beating, DNA extraction of ciliates from soil samples was optimized with a combination of freeze-thaw cycles and ultrasonication. We applied this nested PCR-DGGE method to agricultural soils amended with 0, 120, 300, and 600 t ha⁻¹ year⁻¹

SUBMITTER: Shimano S 

PROVIDER: S-EPMC4036011 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature