Application of nested PCR-DGGE.pdf

http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME11287 Microbes Environ. Vol. 27, No. 2, 136–141, 2012 Application of Nested PCR-DGGE (Denaturing Gradient Gel Electrophoresis) for the Analysis of Ciliate Communities in Soils SATOSHI SHIMANO 1 , MITSUO SAMBE 1 , and YASUHIRO KASAHARA 2 * 1 Miyagi University of Education, Aramaki-aza-Aoba, Aoba-ku, Sendai 980–0845, Japan; and 2 Institute of Low Temperature Science, Hokkaido University, Kita 19, Nishi 8, Kita-ku, Sapporo 060–0819, Japan (Received August 24, 2011—Accepted November 1, 2011—Published online December 1, 2011) 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 of livestock slurry. The results from the DGGE profiles and principal component analysis (PCA) revealed that the supplement of slurry to soils influenced the ciliate communities. From phylogenetic analysis, 108 DGGE bands were assigned to six classes, which included Spirotrichea and Colpodea, of the subphylum Intramacronucleata, and one class of the subphylum Postciliodesmatophora. These results indicated that a wide variety of taxonomic groups were detected by DGGE profiling. Thus, the nested PCR-DGGE method described here could clearly differentiate between ciliate communities within soil samples and allo