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Optimisation of protocol for effective detachment and selective recovery of the representative bacteria for extraction of metagenomic DNA from Eucalyptus spp. woodchips

Authors :
Chika F. Nnadozie
Johnson Lin
Roshini Govinden
Source :
Journal of Microbiological Methods. 148:155-160
Publication Year :
2018
Publisher :
Elsevier BV, 2018.

Abstract

For some environments such as planktonic/aqueous environments, the separation of bacteria cells from eukaryotic cells prior to DNA extraction using filtration is relatively straightforward. However, for woodchips, the bacteria are attached/embedded within the wood matrix, which prevents easy removal of bacterial cells. In this study, a method for the selective extraction of DNA from bacteria inhabiting Eucalyptus spp. woodchips has been developed. The objective was to compare milled and unmilled woodchips processed via three detachment methods, viz., sonication, vortexing and shaking followed by filtration using Teflon filters according to three relevant criteria: DNA yield, DNA purity and quality of DNA. Highest DNA yield was obtained by milling and vortexing for 10 min (77.50 ± 5.17 ng/μl), followed by milling and vortexing for 2 min (61.00 ± 6.56 ng/μl), unmilled and vortexing for 10 min (38.67 ± 5.17 ng/μl) and milled and shaking for 2 h (31.62 ± 5.17 ng/μl). The lowest DNA yield was obtained by using unmilled woodchips and 5 min of sonication treatment (7.00 ± 1.22 ng/μl). There was no significant difference in DNA purity for milled or unmilled woodchips processed via the three detachment methods. Duration of cell detachment treatment did not significantly influence DNA yield and purity. Following optimisation experiments, it was possible to extract bacterial DNA using milled woodchips and 10 minute vortexing devoid of DNA from the host background and other associated eukaryotes and of sufficient quality and quantity for metagenomic analysis.

Details

ISSN :
01677012
Volume :
148
Database :
OpenAIRE
Journal :
Journal of Microbiological Methods
Accession number :
edsair.doi.dedup.....d6b6e18428af1b97f1d19ff0f5dec62e
Full Text :
https://doi.org/10.1016/j.mimet.2018.04.009