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Nitrogen-fixing bacteria are diverse, and most of the known taxa have not yet been cultivated in the laboratory. These organisms enzymatically transform dinitrogen gas from the atmosphere into ammonium equivalents needed for biosynthesis of essential cellular macromolecules. Nitrogen-fixing microorganisms are globally significant in that they provide the only natural biological source of fixed nitrogen in the biosphere. This analysis will be of great utility to those engaged in molecular analysis of nifH genes from isolates and environmental samples.
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Universal primers that performed well in silico were tested empirically with soil samples and with genomic DNA from a phylogenetically diverse set of nitrogen-fixing strains. In addition, many of these primers will amplify genes that do not mediate nitrogen fixation, and thus it would be advisable for researchers to screen their sequencing results for the presence of non-target genes before analysis. The nifH primers we evaluated vary in their phylogenetic bias and their ability to recover sequences from commonly sampled environments. We found that there are 15 universal nifH primers that target 90% or more of nitrogen fixers, but that there are also 23 nifH primers that target less than 50% of nifH sequences. We performed an in silico analysis of the specificity and coverage of 51 universal and 35 group-specific nifH primers by using an aligned database of 23,847 nifH sequences. Numerous PCR primers have been designed to amplify nifH, but a comprehensive evaluation of nifH PCR primers has not been performed. The nifH gene is the most widely sequenced marker gene used to identify nitrogen-fixing Bacteria and Archaea.