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Appl Environ Microbiol. Mar 1993; 59(3): 695–700.
PMCID: PMC202176

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.

Abstract

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

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  • Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol. 1990 Jun;56(6):1919–1925. [PMC free article] [PubMed]
  • Amann RI, Stromley J, Devereux R, Key R, Stahl DA. Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms. Appl Environ Microbiol. 1992 Feb;58(2):614–623. [PMC free article] [PubMed]
  • Børresen AL, Hovig E, Brøgger A. Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene-specific probes. Mutat Res. 1988 Nov;202(1):77–83. [PubMed]
  • Cariello NF, Scott JK, Kat AG, Thilly WG, Keohavong P. Resolution of a missense mutant in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRT Munich. Am J Hum Genet. 1988 May;42(5):726–734. [PMC free article] [PubMed]
  • Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991 Jul 25;19(14):4008–4008. [PMC free article] [PubMed]
  • Fischer SG, Lerman LS. Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. Cell. 1979 Jan;16(1):191–200. [PubMed]
  • Fischer SG, Lerman LS. DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1579–1583. [PMC free article] [PubMed]
  • Giovannoni SJ, Britschgi TB, Moyer CL, Field KG. Genetic diversity in Sargasso Sea bacterioplankton. Nature. 1990 May 3;345(6270):60–63. [PubMed]
  • Giovannoni SJ, Turner S, Olsen GJ, Barns S, Lane DJ, Pace NR. Evolutionary relationships among cyanobacteria and green chloroplasts. J Bacteriol. 1988 Aug;170(8):3584–3592. [PMC free article] [PubMed]
  • Gray M, Charpentier A, Walsh K, Wu P, Bender W. Mapping point mutations in the Drosophila rosy locus using denaturing gradient gel blots. Genetics. 1991 Jan;127(1):139–149. [PMC free article] [PubMed]
  • Lerman LS, Fischer SG, Hurley I, Silverstein K, Lumelsky N. Sequence-determined DNA separations. Annu Rev Biophys Bioeng. 1984;13:399–423. [PubMed]
  • Medlin L, Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene. 1988 Nov 30;71(2):491–499. [PubMed]
  • Myers RM, Fischer SG, Lerman LS, Maniatis T. Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res. 1985 May 10;13(9):3131–3145. [PMC free article] [PubMed]
  • Myers RM, Maniatis T, Lerman LS. Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol. 1987;155:501–527. [PubMed]
  • Neefs JM, Van de Peer Y, Hendriks L, De Wachter R. Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res. 1990 Apr 25;18 (Suppl):2237–2317. [PMC free article] [PubMed]
  • Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. [PubMed]
  • Sheffield VC, Cox DR, Lerman LS, Myers RM. Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci U S A. 1989 Jan;86(1):232–236. [PMC free article] [PubMed]
  • Uitterlinden AG, Slagboom PE, Knook DL, Vijg J. Two-dimensional DNA fingerprinting of human individuals. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2742–2746. [PMC free article] [PubMed]
  • Ward DM, Weller R, Bateson MM. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature. 1990 May 3;345(6270):63–65. [PubMed]
  • Woese CR. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. [PMC free article] [PubMed]

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