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Flotation as a tool for indirect DNA extraction from soil

Skorupa Parachin, Nadia LU ; Schelin, Jenny LU ; Norling, Börje; Rådström, Peter LU and Gorwa-Grauslund, Marie-Francoise LU (2010) In Applied Microbiology and Biotechnology 87(5). p.1927-1933
Abstract
Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a... (More)
Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 mug DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Applied Microbiology and Biotechnology
volume
87
issue
5
pages
1927 - 1933
publisher
Springer
external identifiers
  • wos:000280036300034
  • pmid:20532881
  • scopus:77955558634
ISSN
1432-0614
DOI
10.1007/s00253-010-2691-3
language
English
LU publication?
yes
id
de09396d-c35e-4b35-8e56-fd3418e62ffd (old id 1626259)
date added to LUP
2010-08-23 12:43:02
date last changed
2018-05-29 11:34:00
@article{de09396d-c35e-4b35-8e56-fd3418e62ffd,
  abstract     = {Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 mug DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences.},
  author       = {Skorupa Parachin, Nadia and Schelin, Jenny and Norling, Börje and Rådström, Peter and Gorwa-Grauslund, Marie-Francoise},
  issn         = {1432-0614},
  language     = {eng},
  number       = {5},
  pages        = {1927--1933},
  publisher    = {Springer},
  series       = {Applied Microbiology and Biotechnology},
  title        = {Flotation as a tool for indirect DNA extraction from soil},
  url          = {http://dx.doi.org/10.1007/s00253-010-2691-3},
  volume       = {87},
  year         = {2010},
}