Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences

A defined template mixture of seven closely related 16S-rDNA clones was use d in a PCR-cloning experiment to assess and track sources of artifactual se quence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a m utational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequ ence clusters detected in environmental clone libraries.