Profiling and monitoring of microbial populations by denaturing high-performance liquid chromatography

Abstract

We describe a new molecular technique for the analysis of microbial species and complex microbial populations based on the separation of PCR-amplified 16S rDNA fragments by denaturing high-performance liquid chromatography (DHPLC). Using marine bacterial samples, we determined the optimum conditions for the analysis of bacterial species and the examination of complex bacterial assemblages obtained from different environments. The incorporation of a 40-bp GC clamp into the amplification primer was essential to effectively discriminate genetic differences in DHPLC-primers with a 20-, 10-, or 0-bp GC clamp length were less efficient. A 64.5 degrees C column temperature in DHPLC allowed optimal separation of species in a complex bacterial population. PCR-DHPLC analysis of bacterial assemblages demonstrated profiles with distinguishable peaks, which constituted the different populations and their degree of abundance. Fraction collection and DNA sequencing from profile peaks enabled bacterial identification. PCR-DHPLC analysis can also provide opportunities for describing bacterial communities, cloning bacteria, and monitoring bacterial populations in environments of interest.