DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms

摘要: Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually the form of 13C, 18O, or 15N. After on substrate and subsequent extraction biomarkers, typically nucleic acids proteins, SIP technique used for recovery analysis isotope-labeled biomarkers from populations. In years following initial development DNA- RNA-based SIP, it was common practice characterize labeled targeted gene analysis. Such approaches involved fingerprint-based analyses sequencing clone libraries containing 16S rRNA genes functional marker amplicons. Although molecular fingerprinting remains a valuable approach rapid confirmation isotope labeling, recent advances technology mean that possible obtain affordable comprehensive amplicon profiles, metagenomes, metatranscriptomes experiments. Not only can abundance groups be inferred but bin, assemble, explore individual genomes build hypotheses about metabolic capabilities microorganisms. Analysis mRNA more advance provide independent metatranscriptome-based The power metatranscriptomics often correlates closely with corresponding activity encoded enzymes, thus insight into metabolism at time sampling. Together, these have improved sensitivity methods allow use ecologically relevant concentrations. Particularly as improve costs continue drop, we expect integration multiple omics-based will become prevalent components ecology studies, leading further breakthroughs our understanding novel elucidation function communities. this chapter protocols obtaining DNA, RNA, proteins downstream analyses.