Caister Academic Press

Quantification of Functional Microbial Nitrogen Cycle Genes in Environmental Samples

David Correa-Galeote, Germán Tortosa and Eulogio J. Bedmar
from: Metagenomics of the Microbial Nitrogen Cycle: Theory, Methods and Applications (Edited by: Diana Marco). Caister Academic Press, U.K. (2014)

Abstract

The nitrogen (N) cycle comprises a large number of oxidative and reductive reactions that are catalyzed by wide variety of enzymes. Genes coding for most of the N-cycle enzymes have been shown to be present in a diverse polyphyletic group of microorganisms, including bacteria, archaea and fungi. Therefore, a 16S rRNA phylogeny-based approach to study those microbial populations is not possible. Because cultivation-dependent methods are selective for certain microorganisms, molecular methods have been developed to study the ecology and to assess abundance and diversity composition of nitrogen cycling microorganisms in environmental samples. DNA extraction followed by PCR amplification of genes that encode key functional enzymes of the N-cycle are used to study which genes and/or phylotypes are functionally important in the environment. Methods for DNA isolation and purification from environmental samples will be addressed whilst considering the main functional gene targets used to study the nitrogen fixation, nitrification and denitrification processes within the nitrogen cycle. The fluorescence-based quantitative real-time polymerase chain reaction (qPCR) has proven useful for quantification of nucleic acids in samples obtained from numerous diverse sources. Here, we describe relevant experimental conditions for utilization of qPCR to quantify the 16S rRNA, amoA and nar/nap, nirK/nirS, c-nor/q-nor and nos genes that encode synthesis of key enzymes involved in redox reactions of the N-cycle read more ...
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