Open AccessHigh-Sensitivity Stable-Isotope Probing by a Quantitative Terminal Restriction Fragment Length Polymorphism Protocol
Author(s) -
Peter F. Andeer,
Stuart E. Strand,
David A. Stahl
Publication year - 2012
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.05973-11
Subject(s) - terminal restriction fragment length polymorphism , biology , restriction fragment length polymorphism , stable isotope probing , microbacterium , amplicon , genomic dna , escherichia coli , genetics , restriction fragment , dna , polymerase chain reaction , microbiology and biotechnology , 16s ribosomal rna , bacteria , gene , microorganism
Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)–TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli ,Rhodococcus ,Variovorax , andMicrobacterium ), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.