Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil

Applied Soil Ecology : a Section of Agriculture, Ecosystems & Environment
Andreas O WagnerPaul Illmer

Abstract

Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol-chloroform-isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of t...Continue Reading

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Citations

Nov 10, 2015·Journal of Applied Microbiology·Nicoletta Pasqualina MangiaPietrino Deiana
Mar 24, 2016·Antonie van Leeuwenhoek·Christoph ReitschulerPaul Illmer
Jun 10, 2015·Journal of Microbiological Methods·Glade DlottStephanie Yarwood
Jul 14, 2016·Microbial Ecology·Milko A JorqueraMaría de La Luz Mora
Dec 20, 2016·Nature Microbiology·Paul CariniNoah Fierer
Feb 10, 2018·Applied Microbiology and Biotechnology·Magdalena NaglerJudith Ascher-Jenull
Apr 11, 2020·Archives of Microbiology·Elisandra Triches da CunhaAna Carolina Maisonnave Arisi
Jul 14, 2020·GigaScience·Fangming YangHuanzi Zhong
May 15, 2021·Frontiers in Microbiology·Maraike ProbstMaría Gómez-Brandón

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Methods Mentioned

BETA
PMA
PCR
electrophoresis

Software Mentioned

Gene
Rotor
StatSoft
GelCompar
Statistica
SigmaPlot

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