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J Microbiol Methods. 2012 Jan;88(1):35-40. doi: 10.1016/j.mimet.2011.10.007. Epub 2011 Oct 26.

Nonlinear electrophoresis for purification of soil DNA for metagenomics.

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1
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada N2L 3G1.

Abstract

Purification of microbial DNA from soil is challenging due to the co-extraction of humic acids and associated phenolic compounds that inhibit subsequent cloning, amplification or sequencing. Removal of these contaminants is critical for the success of metagenomic library construction and high-throughput sequencing of extracted DNA. Using three different composite soil samples, we compared a novel DNA purification technique using nonlinear electrophoresis on the synchronous coefficient of drag alteration (SCODA) instrument with alternate purification methods such as direct current (DC) agarose gel electrophoresis followed by gel filtration or anion exchange chromatography, Wizard DNA Clean-Up System, and the PowerSoil DNA Isolation kit. Both nonlinear and DC electrophoresis were effective at retrieving high-molecular weight DNA with high purity, suitable for construction of large-insert libraries. The PowerSoil DNA Isolation kit and the nonlinear electrophoresis had high recovery of high purity DNA suitable for sequencing purposes. All methods demonstrated high consistency in the bacterial community profiles generated from the DNA extracts. Nonlinear electrophoresis using the SCODA instrument was the ideal methodology for the preparation of soil DNA samples suitable for both high-throughput sequencing and large-insert cloning applications.

PMID:
22056233
DOI:
10.1016/j.mimet.2011.10.007
[Indexed for MEDLINE]
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