Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria.

The 16S rDNA genes of an apparently pure culture of a psychrophilic and strict barophilic bacterium (WHB 46) were studied by PCR-mediated amplification and cloning into phage M13 mp18. Sequence analysis of five individual clones revealed the presence of two different 16S rDNA types. The homology value of 90% indicates that culture WHB 46 is actually composed of two closely related species (WHB 46-1 and 46-2). Both strains are members of the γ-subdivision of proteobacteria. Analysis of a sixth clone (WHB 46-1/2) leads to the conclusion that it represents a 16S rDNA hybrid molecule assembled during the PCR reaction. This hypothesis was confirmed by secondary structure analysis of the chimeric rDNA. The appearance of such hybrid molecules point to a potential risk in studies on the diversity of bacterial populations by analysis of rDNA pattern via PCR-mediated amplification because they suggest the existence of organisms that do not actually exist in the sample investigated.