Nitrogenase genes in PCR and RT-PCR reagents: implications for studies of diversity of functional genes

Studies of the diversity of microorganisms in the environment have been facilitated by use of PCR and reverse transcription PCR (RT-PCR). Inhibition of the PCR by complex sample matrices and low abundance of some target microorganisms require the use of high-sensitivity amplification procedures, involving a large number of cycles or nested PCR methods. Using these methods, we frequently observed contamination of the amplification reagents, including polymerases, by genomic DNA containing nitrogenase (nifH) and rRNA genes. Contaminating genes were sequenced and found to belong to a variety of rRNA clades, but only three major nifH clades. These sequence types included a few nifH sequences reported in previous studies of the environment. Contamination could be reduced by restriction digestion and ultrafiltration of PCR reagents, but efficiency of amplification was also reduced. Our results suggest that studies relying on large numbers of PCR amplification cycles to assess environmental gene diversity should take precautions to assure that clone libraries generated from amplified PCR products are not the result of contaminated PCR reagents.