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Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation

Courtois, S; Frostegård, Å; Göransson, Pernilla LU ; Depret, G; Jeannin, P and Simonet, P (2001) In Environmental Microbiology 3(7). p.431-439
Abstract
All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S... (More)
All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S rRNA gene fragment, followed by hybridization of the amplified fragments to a set of specific probes to assess the phylogenetic diversity of our samples. Control parameters, such as the relationship between amount of DNA template and amount of PCR product and the influence of competing DNA on PCR amplification, were first examined. Comparison between extraction methods showed that less DNA was extracted when cells were first separated from the soil matrix (0.4 µg g1 dry weight soil versus 38–93 µg g1 obtained by in situ lysis methods). However, with the exception of the γ-subclass of Proteobacteria, there was no significant difference in the spectrum of diversity resulting from the two extraction strategies. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Environmental Microbiology
volume
3
issue
7
pages
431 - 439
publisher
Wiley-Blackwell
external identifiers
  • scopus:0035206784
ISSN
1462-2920
DOI
10.1046/j.1462-2920.2001.00208.x
language
English
LU publication?
yes
id
0538ee71-528e-4b0b-8f3b-37098c4ea072 (old id 149409)
date added to LUP
2007-06-29 09:26:27
date last changed
2018-01-07 06:20:14
@article{0538ee71-528e-4b0b-8f3b-37098c4ea072,
  abstract     = {All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S rRNA gene fragment, followed by hybridization of the amplified fragments to a set of specific probes to assess the phylogenetic diversity of our samples. Control parameters, such as the relationship between amount of DNA template and amount of PCR product and the influence of competing DNA on PCR amplification, were first examined. Comparison between extraction methods showed that less DNA was extracted when cells were first separated from the soil matrix (0.4 µg g1 dry weight soil versus 38–93 µg g1 obtained by in situ lysis methods). However, with the exception of the γ-subclass of Proteobacteria, there was no significant difference in the spectrum of diversity resulting from the two extraction strategies.},
  author       = {Courtois, S and Frostegård, Å and Göransson, Pernilla and Depret, G and Jeannin, P and Simonet, P},
  issn         = {1462-2920},
  language     = {eng},
  number       = {7},
  pages        = {431--439},
  publisher    = {Wiley-Blackwell},
  series       = {Environmental Microbiology},
  title        = {Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation},
  url          = {http://dx.doi.org/10.1046/j.1462-2920.2001.00208.x},
  volume       = {3},
  year         = {2001},
}