Nested PCR bias: a case study of Pseudomonas spp. in soil microcosms.

Nested PCR methods combined with denaturing gradient gel electrophoresis (DGGE) are widely used for the detection of low copy number species or for the analysis of group-specific community profiles. With an appropriate number of PCR cycles during the first round of amplification, initial differences in the copy numbers of different DNA fragments that are targeted can be maintained during the second round without significant bias. However, if an excessive number of cycles in used in the first round, relative differences in the copy numbers of the targeted sequences can be obscured. Here we demonstrate the effect of "nested PCR bias" in a case study with PCR-DGGE of 16S rRNA gene sequences targeting Pseudomonas spp. following exposure of soil to naphthalene vapors. Our results demonstrate artifacts caused by nested PCR bias can be substantially minimized by calibrating the number of first round PCR cycles, thereby preserving the ability to obtain semiquantitative data for evaluating changes in gene copy numbers over time.