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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Food Safety and Enteric Pathogens Research » Research » Publications at this Location » Publication #182151


item Scupham, Alexandra
item Jones, Jennifer
item Wesley, Irene

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/8/2006
Publication Date: 2/20/2007
Citation: Scupham, A.J., Jones, J., Wesley, I.V. 2007. Comparison of DNA extraction methods for analysis of turkey cecal microbiota. Journal of Applied Microbiology. 102(2):401-409.

Interpretive Summary: Molecular analysis of microbial diversity from complex ecosystems is a field that has developed in the last 20 years as an alternative to classical culturing techniques. Molecular methods successfully identified a third kingdom of life, the Archea, and revealed that the bacterial diversity on the planet is far greater than originally believed. These methods have been used to analyze ecosystems such as soil, seawater, biofilms and feces, and allowed initial investigations into functional relationships between the organisms identified therein. However, these investigations are dependent upon a few critical steps, primarily the isolation and purification of DNA from environmental samples. In this work we compared 14 methods for isolating DNA from turkey cecal contents. We found a range of quantity and purity of the final products, with the Invitrogen Easy DNA kit generating the greatest DNA yield. Using a community fingerprinting technique we measured the number of bacterial and fungal species identified by each DNA extraction method and found that each method identified a unique subset of the microbial community. The presence of contaminants inhibiting amplification of ribosomal sequences is likely responsible for this effect. Finally, this work indicates a complex fungal community resides in the poultry intestine. Our work indicates that current molecular techniques are identifying only part of the microbial diversity present in complex samples. Methods for enhancing retention of DNA while removing contaminants need to be refined, and researchers need to carefully match DNA extraction methods to their sample types.

Technical Abstract: To compare fourteen DNA isolation protocols from eight commercially available kits, turkey cecal microbial diversity was evaluated. Kits included Qbiogene Fast DNA, Qiagen QIAamp DNA Stool, MoBio Ultra Clean Fecal, MoBio Ultra Clean Soil, Roche High Pure, Epicentre Soilmaster, BioRad Aquapure DNA and Invitrogen Easy DNA. DNA quantity and quality were assessed and competitive PCR was used to quantify the 16S bacterial rRNA genes. The Invitrogen Easy-DNA Kit, method I4, yielded over eight times more DNA than any other method (3144 +/- 873 ug/gram of sample, P < 0.05). Considerable DNA shearing was observed in preparations using the Qbiogene FastDNA Kit method QY, Invitrogen Easy-DNA Kit method I3 and the Epicentre Soilmaster DNA Extraction Kit, method ES. The MoBio Ultra Clean Soil kit produced 1.2 x 10**13 bacterial 16S DNA copies per gram of sample; 1.5 times more than the next greatest yield. Bacterial and fungal species richness was estimated by Automated Ribosomal Intergenic Spacer Analysis (ARISA). The Invitrogen Easy-DNA Kit, method I4, generated the greatest bacterial species richness (46 +/- 7 peaks) while the Qbiogene FastDNA with Boom L6 lysis buffer generated the highest fungal species richness (77 +/- 4 peaks). Cluster analysis indicated different DNA extraction methods reproducibly generated different microbial community composition. These results indicate experimental design for molecular ecology studies should include careful selection of DNA extraction method.