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Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study

Abstract

Based on the comparative study of the DNA extracts from two soil samples obtained by three commercial DNA extraction kits, we evaluated the influence of the DNA quantity and purity indices (the absorbance ratios A260/280 and A260/230, as well as the absorbance value A320 indicating the amount of humic substances) on polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and a functional gene microarray used in the study of microbial communities. Numbers and intensities of the DGGE bands are more affected by the A260/280 and A320 values than by the ratio A260/230 and conditionally affected by the DNA yield. Moreover, we demonstrated that the DGGE band pattern was also affected by the preferential extraction due to chemical agents applied in the extraction. Unlike DGGE, microarray is more affected by the A260/230 and A320 values. Until now, the successful PCR performance is the mostly used criterion for soil DNA purity. However, since PCR was more influenced by the A260/280 ratio than by A260/230, it is not accurate enough any more for microbial community assessed by non-PCR-based methods such as microarray. This study provides some useful hints on how to choose effective DNA extraction method for the subsequent assessment of microbial community.

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Acknowledgements

The manuscript is a joint approach within the working group “Biodiversity” of the Helmholtz Association, Germany. The authors would like to thank Eva Mareike Seeger, Mareike Braeckevelt, and Heidrun Paschke in the group of the Department of Bioremediation (UFZ, Germany) for the help during the sampling at the SAFIRA plant in Bitterfeld and the group in the Institute for Environmental Genomics (Oklahoma, USA) for providing the microarray chips, as well as Dr. Naixin Li for the valuable discussion. The construction of microarray chips and its functional gene array predecessors is supported by the Environmental Stress Pathway Project (ESPP) of the Virtual Institute for Microbial Stress and Survival (http://vimss.lbl.gov) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomics: GTL Program, through contract DE-AC02-05CH11231 with Lawrence Berkeley National Laboratory.

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Correspondence to Jing Ning or Jost Liebich.

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Ning, J., Liebich, J., Kästner, M. et al. Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study. Appl Microbiol Biotechnol 82, 983–993 (2009). https://doi.org/10.1007/s00253-009-1912-0

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Keywords

  • DNA purity indices
  • DNA yield
  • DGGE
  • Functional gene microarray
  • Microbial community analyses