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Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics

Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics ▿ † Marketa Sagova-Mareckova 1 , * , Ladislav Cermak 1 , Jitka Novotna 1 , Kamila Plhackova 1 , Jana Forstova 2 and Jan Kopecky 1 1 Laboratory for Biology of Secondary Metabolism, Institute of Microbiology of the ASCR v.v.i., Videnska 1083, CZ-14220 Prague 4, Czech Republic 2 Institute of Applied Mathematics and Information Technologies, Faculty of Sciences, Charles University in Prague, Albertov 6, CZ-12843 Prague 2, Czech Republic ABSTRACT Seven methods of soil DNA extraction and purification were tested in a set of 14 soils differing in bedrock, texture, pH, salinity, moisture, organic matter content, and vegetation cover. The methods introduced in this study included pretreatment of soil with CaCO 3 or purification of extracted DNA by CaCl 2 . The performance of innovated methods was compared to that of the commercial kit Mo Bio PowerSoil and the phenol-chloroform-based method of D. N. Miller, J. E. Bryant, E. L. Madsen, and W. C. Ghiorse (Appl. Environ. Microbiol. 65:4715-4724, 1999). This study demonstrated significant differences between the tested methods in terms of DNA yield, PCR performance, and recovered bacterial diversity. The differences in DNA yields were correlated to vegetation cover, soil pH, and clay content. The differences in PCR performances were correlated to vegetation cover and soil pH. The innovative methods improved PCR performance in our set of soils, in particular for forest acidic soils. PCR was successful in 95% of cases by the method using CaCl 2 purification and in 93% of cases by the method based on CaCO 3 pretreatment, but only in 79% by Mo Bio PowerSoil, for our range of soils. Also, the innovative methods recovered a higher percentage of actinomycete diversity from a subset of three soils. Recommendations include the assessment of soil characteristics prior to selecting the optimal protocol for soil DNA extraction and purification. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied and Environmental Microbiology American Society For Microbiology

Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics

Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics

Applied and Environmental Microbiology , Volume 74 (9): 2902 – May 1, 2008

Abstract

Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics ▿ † Marketa Sagova-Mareckova 1 , * , Ladislav Cermak 1 , Jitka Novotna 1 , Kamila Plhackova 1 , Jana Forstova 2 and Jan Kopecky 1 1 Laboratory for Biology of Secondary Metabolism, Institute of Microbiology of the ASCR v.v.i., Videnska 1083, CZ-14220 Prague 4, Czech Republic 2 Institute of Applied Mathematics and Information Technologies, Faculty of Sciences, Charles University in Prague, Albertov 6, CZ-12843 Prague 2, Czech Republic ABSTRACT Seven methods of soil DNA extraction and purification were tested in a set of 14 soils differing in bedrock, texture, pH, salinity, moisture, organic matter content, and vegetation cover. The methods introduced in this study included pretreatment of soil with CaCO 3 or purification of extracted DNA by CaCl 2 . The performance of innovated methods was compared to that of the commercial kit Mo Bio PowerSoil and the phenol-chloroform-based method of D. N. Miller, J. E. Bryant, E. L. Madsen, and W. C. Ghiorse (Appl. Environ. Microbiol. 65:4715-4724, 1999). This study demonstrated significant differences between the tested methods in terms of DNA yield, PCR performance, and recovered bacterial diversity. The differences in DNA yields were correlated to vegetation cover, soil pH, and clay content. The differences in PCR performances were correlated to vegetation cover and soil pH. The innovative methods improved PCR performance in our set of soils, in particular for forest acidic soils. PCR was successful in 95% of cases by the method using CaCl 2 purification and in 93% of cases by the method based on CaCO 3 pretreatment, but only in 79% by Mo Bio PowerSoil, for our range of soils. Also, the innovative methods recovered a higher percentage of actinomycete diversity from a subset of three soils. Recommendations include the assessment of soil characteristics prior to selecting the optimal protocol for soil DNA extraction and purification.

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References (34)

Publisher
American Society For Microbiology
Copyright
Copyright © 2008 by the American society for Microbiology.
ISSN
0099-2240
eISSN
1098-5336
DOI
10.1128/AEM.02161-07
pmid
18344341
Publisher site
See Article on Publisher Site

Abstract

Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics ▿ † Marketa Sagova-Mareckova 1 , * , Ladislav Cermak 1 , Jitka Novotna 1 , Kamila Plhackova 1 , Jana Forstova 2 and Jan Kopecky 1 1 Laboratory for Biology of Secondary Metabolism, Institute of Microbiology of the ASCR v.v.i., Videnska 1083, CZ-14220 Prague 4, Czech Republic 2 Institute of Applied Mathematics and Information Technologies, Faculty of Sciences, Charles University in Prague, Albertov 6, CZ-12843 Prague 2, Czech Republic ABSTRACT Seven methods of soil DNA extraction and purification were tested in a set of 14 soils differing in bedrock, texture, pH, salinity, moisture, organic matter content, and vegetation cover. The methods introduced in this study included pretreatment of soil with CaCO 3 or purification of extracted DNA by CaCl 2 . The performance of innovated methods was compared to that of the commercial kit Mo Bio PowerSoil and the phenol-chloroform-based method of D. N. Miller, J. E. Bryant, E. L. Madsen, and W. C. Ghiorse (Appl. Environ. Microbiol. 65:4715-4724, 1999). This study demonstrated significant differences between the tested methods in terms of DNA yield, PCR performance, and recovered bacterial diversity. The differences in DNA yields were correlated to vegetation cover, soil pH, and clay content. The differences in PCR performances were correlated to vegetation cover and soil pH. The innovative methods improved PCR performance in our set of soils, in particular for forest acidic soils. PCR was successful in 95% of cases by the method using CaCl 2 purification and in 93% of cases by the method based on CaCO 3 pretreatment, but only in 79% by Mo Bio PowerSoil, for our range of soils. Also, the innovative methods recovered a higher percentage of actinomycete diversity from a subset of three soils. Recommendations include the assessment of soil characteristics prior to selecting the optimal protocol for soil DNA extraction and purification.

Journal

Applied and Environmental MicrobiologyAmerican Society For Microbiology

Published: May 1, 2008

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