Evaluation of DNA extraction methods for molecular analyses of microbial communities in modern calcareous microbialites

Brian D. Wade, Ferran Garcia-Pichel

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

We evaluated and optimized three rapid methods for extraction of high-quality DNA from carbonate-encrusted microbial communities using modern calcifying oncolites built by cyanobacteria and diatoms in a high-calcium freshwater river. Pulverization, acid (HCl) dissolution, and chelator-mediated (EDTA) dissolution of the carbonate matrix were used and optimized to liberate microbial cells from their mineral encasing. This was followed by cell lysis and DNA extraction and isolation. HCl dissolution yielded no measurable DNA, probably due to hydrolysis, whereas pulverization and EDTA dissolution yielded averages of 3.5 and 7.8 μg per gram of sample, respectively, of high molecular weight DNA. These DNA isolates could be used successfully for PCR-amplification of 16S rRNA gene segments (alleles) and subsequent fingerprinting of the cyanobacterial (including diatoms) and total bacterial communities through denaturing gradient gel electrophesis (DGGE) separation. Fingerprints showed no differences in microbial community composition between the pulverization and EDTA dissolution methods. While the pulverization method was faster, we demonstrate here that EDTA carbonate dissolution is superior because it preserves vertical stratification of the microbial communities lost using the pulverization method, thus allowing for spatially resolved community analyses.

Original languageEnglish (US)
Pages (from-to)549-561
Number of pages13
JournalGeomicrobiology Journal
Volume20
Issue number6
StatePublished - Nov 2003

Fingerprint

extraction method
microbial community
Dissolution
Edetic Acid
dissolution
Carbonates
EDTA
DNA
Diatoms
diatom
Dermatoglyphics
Cyanobacteria
carbonate
Chelating Agents
Fresh Water
rRNA Genes
Rivers
lysis
Minerals
Hydrolysis

Keywords

  • Bacteria
  • Calcifying microbialites
  • Cyanobacteria
  • Denaturing gradient gel electrophoresis (DGGE)
  • DNA extraction
  • Laminated microbial communities
  • Molecular analyses
  • Oncolites
  • Spatial resolution
  • Stromatolites
  • Thrombolites

ASJC Scopus subject areas

  • Earth and Planetary Sciences (miscellaneous)
  • Microbiology
  • Environmental Science(all)
  • Environmental Chemistry

Cite this

Evaluation of DNA extraction methods for molecular analyses of microbial communities in modern calcareous microbialites. / Wade, Brian D.; Garcia-Pichel, Ferran.

In: Geomicrobiology Journal, Vol. 20, No. 6, 11.2003, p. 549-561.

Research output: Contribution to journalArticle

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