Evaluation of the denaturing gradient gel electrophoresis-apparatus as a parameter influencing soil microbial community fingerprinting
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We compared two denaturing gradient gel electrophoresis (DGGE) systems—DCode (Biorad, Hercules, CA, USA) and PhorU (Ingeny, Leiden, NL), performing community level 16S and 18S rRNA gene fragment-PCR-DGGE with total DNA extracted from upland pasture soil used for outdoor cattle husbandry. The methodological evaluation of the DGGE apparatus as parameter influencing DGGE fingerprinting, based on cluster analysis of soil bacterial and fungal community fingerprints, was made in terms of the resulting information about microbial community structures and their response to different degrees of cattle impact. Although the comparative DGGE analysis with different DGGE systems provided similar clustering of microbial community structures in correlation with the degree of cattle impact, our results suggest the DGGE system to be a factor influencing DGGE analysis. To our knowledge this is the first attempt to investigate the hypothetical impact of the DGGE system due to different technical characteristics, recommending the use of one and the same DGGE apparatus throughout an experiment, if the monitoring of microbial community structures requires multiple gel-to-gel analysis.
KeywordsCattle impact DGGE DGGE interfering factors Soil microbial community fingerprinting
The study was realized within the scientific cooperation between the Consiglio Nazionale di Ricerca, Italy, and the Academy of Sciences of the Czech Republic. We are grateful to the Ente Cassa di Risparmio di Firenze, the Ministry of Education of the Czech Republic (LC06066) and the Grant Agency of the Academy of Sciences of the Czech Republic (IAA600660605) for financial support. The authors are grateful for useful advice and suggestions from Dr. F. Valori and the anonymous referees.
- Ascher J, Ceccherini MT, Landi L, Mench M, Pietramellara G, Nannipieri P, Renella G (2009a) Composition, biomass and activity of microflora, and leaf yields and foliar elemental concentrations of lettuce, after in situ stabilization of an arsenic-contaminated soil. Appl Soil Ecol 41:351–359CrossRefGoogle Scholar
- Green SJ (2006) A guide to denaturing gradient gel electrophoresis. Online publication. http://ddgehelp.blogspot.com/
- Jirout J, Tříska J, Růžičková K, Elhottová D (2009) Disturbing impact of outdoor cattle husbandry on community of arbuscular mycorrhizal fungi in upland pasture soil. Commun Soil Sci Plant Anal 40:736–745Google Scholar
- Marzorati M, Wittebolle L, Boon N, Daffonchio D, Verstraete W (2008) How to get more out of molecular fingerprints: practical tools for microbial ecology. Environ Microbiol 10:1571–1581Google Scholar
- Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700Google Scholar
- Nübel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann RI, Ludwig W, Backhaus H (1996) Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178:5636–5643Google Scholar
- Renella G, Landi L, Ascher J, Ceccherini MT, Pietramellara G, Mench M, Nannipieri P (2008) Long-term effects of aided phytostabilization of trace elements on microbial biomass and activity, enzyme activities and composition of microbial community in the Jales contaminated mine spoils. Environ Pollut 152:702–712CrossRefGoogle Scholar
- Tebbe CC, Vahjen W (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl Environ Microbiol 59:2657–2665Google Scholar