Comparison of pmoA PCR primer sets as tools for investigating methanotrophdiversity in three Danish soils

Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A6 50, and A189-mb661) were investigated for their ability to assess methanotr oph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable of oxidizing atmospheric concentrations of methane. Ea ch PCR primer set was used to construct a library containing 50 clones from each soil type. The clones from each library were grouped by restriction f ragment length polymorphism, and representatives from each group were seque nced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated by amoA-related sequences or nonspecific PCR products with n onsense open reading frames. The primer set could not be used to assess met hanotroph diversity in these soils. A new pmoA-specific primer, A650, was d esigned in this study. The A189-A650 primer set demonstrated distinct biase s both in clone library analysis and when incorporated into denaturing grad ient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrat ed the largest retrieval of methanotroph diversity of all of the primer set s. However, this primer set did not retrieve sequences linked with novel hi gh-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer set. A combination of all three primer sets appe ars to be required to examine both methanotroph diversity and the presence of novel methane monooxygenase sequences.