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Comparative Study
. 2017 Dec;143:78-86.
doi: 10.1016/j.mimet.2017.10.007. Epub 2017 Oct 19.

Comparison of Ten Different DNA Extraction Procedures With Respect to Their Suitability for Environmental Samples

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Comparative Study

Comparison of Ten Different DNA Extraction Procedures With Respect to Their Suitability for Environmental Samples

Ramona Kuhn et al. J Microbiol Methods. .

Abstract

DNA extraction for molecular biological applications usually requires target optimized extraction procedures depending on the origin of the samples. For environmental samples, a range of different procedures has been developed. We compared the applicability and efficiency of ten selected DNA extraction methods published in recent literature using four different environmental samples namely: activated sludge from a domestic wastewater treatment plant, river sediment, anaerobic digestion sludge and nitrifying enrichment culture. We assessed the suitability of the extraction procedures based on both DNA yield and quality. DNA quantification was performed by both ultra violet (UV) spectrophotometry and fluorescence spectrophotometry after staining with PicoGreen. In our study, DNA yields based on UV measurement were overestimated in most cases while DNA yields from fluorescence measurements correlated well with the sample load on agarose gels of crude DNA. The quality of the DNA extracts was determined by gel electrophoresis of crude DNA and PCR products from 16S rDNA with the universal primer set 27f/1525r. It was observed that gel electrophoresis of crude DNA was not always suitable to evaluate DNA integrity and purity since interfering background substances (e.g. humic substances) were not visible. Therefore, we strongly recommend examining the DNA quality of both crude DNA and 16S rDNA PCR products by gel electrophoresis when a new extraction method is established. Summarizing, we found four out of ten extraction procedures being applicable to all tested samples without noticeable restrictions. The procedure G (according to the standard method 432_10401 of the Lower Saxony State Office for Consumer Protection and Food Safety) had the broadest application range over procedure J (published by Wilson, 2001). These were followed by procedures F (Singka et al., 2012) and A (Bourrain et al., 1999). All four extraction procedures delivered reliable and reproducible crude DNA and PCR products. From an economical point of view, all procedures tested during this study were cheaper compared to commercial DNA extraction kits.

Keywords: DNA extraction; DNA quantification; DNA yield; Environmental samples; Gel electrophoresis; PicoGreen.

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