Denaturing gradient gel electrophoresis polymorphism for rapid 16S rDNA clone screening and microbial diversity study

Abstract

A method based on denaturing gradient gel electrophoresis (DGGE) banding polymorphism was developed for rapid small-subunit (SSU) rDNA clone screening and microbial diversity estimation. Clones with correct rDNA insertion were initially identified using whole-cell PCR with vector-specific primers. Subsequently, the correct PCR products were re-amplified with nested primers in a DGGE analysis. An average of five to 10 individual DGGE-PCR products could be mixed together and loaded into a DGGE well, and at least 125 to 250 clones could be screened and compared within one DGGE gel. This approach eliminated the reproducibility problem associated with DGGE, increased the ability to screen a large set of clones at a relatively affordable cost, and was shown to be more sensitive than the restriction fragment length polymorphism method in identifying 10 different but phylogenetically closely related 16S rDNA fragments from 126 clones obtained from an anaerobic sludge sample.