DNA-Based Stable Isotope Probing.

Microbiomes on Earth are often considered the most heterogeneous biological entities, but their vital roles in driving global biogeochemical cycles often remain elusive. DNA-based stable isotope probing (DNA-SIP) provides a powerful means to establish a direct link between biogeochemical processes and the taxonomic identities of active microorganisms involved in the processes. Combined with high-throughput sequencing, it significantly aids in deciphering ecophysiological functions of active microorganisms at the level of microbial communities. DNA-SIP relies solely on the propagation of targeted microbial communities, during which the entire genomes of daughter cells are synthesized and increasingly C-labeled. This growth on C-labeled substrate in association with cell division provides solid evidence for the functional importance and metabolic potential of targeted microorganisms. The essential prerequisite for a successful DNA-SIP experiment is the identification, with confidence, of isotopically enriched C-DNA, of which the amount is generally too low to allow for the direct measurement of C atomic percent of nucleic acid. The C labeling can be readily identified in the fractionated DNA by quantification of functional genes specific to the known targeted microorganisms, and by high-throughput sequencing of the total microbial communities via 16S rRNA genes without prior knowledge of which microorganisms are C-labeled (i.e., highly enriched in the heavy fractions relative to C (natural isotope abundance) control treatments). In this chapter, the protocol for obtaining DNA highly enriched in heavy isotope is presented using diazotrophic methanotrophs in a paddy soil as a case study.