Metagenomic Analysis of Isotopically Enriched DNA

This detailed protocol describes an approach for combining DNA stable-isotope probing-based enrichment, multiple displacement amplification (MDA), and metagenomics. Together, these three methodologies enable selective access to the genomes of uncultivated organisms that actively grow using isotopically labelled carbon and nitrogen sources. Incubations with stable-isotope-labelled substrates enrich isotopically labelled DNA from functionally relevant micro-organisms; this serves as a filter to reduce the complexity of the metagenome. The MDA step generates sufficient DNA from labelled nucleic acid for metagenomic library construction. Subsequently, genome fragments can be subjected to a variety of screens for phylogenetic or functional genes relevant to active community members. The MDA-generated DNA can also serve as template for direct high-throughput sequencing to aid reconstruction of metabolic pathways of those active organisms. Recent proof-of-concept studies have demonstrated that this novel combination of molecular methods can offer substantial enhancements to gene detection frequencies and may have great future potential for the discovery of novel genes, enzymes, and metabolic pathways.