Authors: Nádia Skorupa Parachin Jenny Schelin Börje Norling Peter Rådström Marie F GorwaGrauslund
Publish Date: 2010/06/09
Volume: 87, Issue: 5, Pages: 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 coextraction of enzymeinhibitory 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 enzymeinhibitory substances by flotation A discontinuous density gradient was designed using a colloidal solution of nontoxic 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 35 μg DNA/g soil and neither PCR nor restriction enzyme digestion of DNA were inhibited Furthermore specific primers enabled recovery of both prokaryotic and eukaryotic sequencesBirgit Johansson is acknowledged for providing fresh soil early morning The BactXtractor medium was kindly provided by FertiPro Belgium This work was financed by the Swedish Research Council VR the European Union project BIOTRACER FOOD2006CT036272 and the Swedish Research Council for Environment Agricultural Sciences and Spatial Planning
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