Single-stranded conformational polymorphism for separation of mixed rRNAS (rRNA-SSCP): a new method for profiling microbial communities.
We show that non-denaturing gel electrophoresis, or single-stranded conformational polymorphism (SSCP), can be used to separate mixtures of full-length rRNAs. Individual bands can then be excised for identification by RT-PCR and sequencing. This has the advantage over profiling methods such as DGGE and T-RFLP that no PCR amplification is involved prior to sequencing; thus, extraction biases aside, it should yield a quantitative picture of community composition in terms of ribosome content. To simplify banding patterns, RNA subsamples (e.g. bacterial 16S rRNA) can first be isolated by magnetic bead capture hybridization. Alternatively, oligonucleotide-directed ribonuclease H (RNase H) digestion can be used to identify bands of interest by running digested samples in parallel to undigested ones. We illustrate the use of this technique to identify a potentially predominant species in a hypersaline microbial mat. We anticipate that rRNA-SSCP will be useful for community profiling; for clone library construction by directed cloning of individual rRNAs; and for following incorporation of radiolabeled substrates at the species level, by gel autoradiography, without advance information or guesswork about which species might be active and abundant.
MacGregor
Barbara J
BJ
Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, Bremen, Germany. bmacgreg@unc.edu
Amann
Rudolf
R
D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000254:growth & development; D017753:Ecosystem; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005618:Fresh Water / Q000382:microbiology*; D019015:Geologic Sediments / Q000382:microbiology*; D008969:Molecular Sequence Data; D018807:Polymorphism, Single-Stranded Conformational; D012335:RNA, Ribosomal / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride; D015003:Yeasts / Q000145:classification / Q000235:genetics / Q000254:growth & development
D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012337:RNA, Ribosomal, 18S; D012965:Sodium Chloride