Evaluation of DNA Extraction Methods for Molecular Analyses of Microbial Communities in Modern Calcareous Microbialites
Titel:
Evaluation of DNA Extraction Methods for Molecular Analyses of Microbial Communities in Modern Calcareous Microbialites
Auteur:
Wade, Brian D. Garcia-Pichel, Ferran
Verschenen in:
Geomicrobiology journal
Paginering:
Jaargang 20 (2003) nr. 6 pagina's 549-561
Jaar:
2003-11
Inhoud:
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 electrophoresis (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.
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