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United States Department of Agriculture

Agricultural Research Service

Title: Extraction of Total Microbial DNA from the Ceceal Contents of Turkeys: Comparison of Methods

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

Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: September 9, 2004
Publication Date: November 14, 2004
Citation: Scupham, A.J., Wesley, I.V. 2004. Extraction of total microbial DNA from the ceceal contents of turkeys: comparison of methods [abstract]. Conference of Research Workers in Animal Diseases. p. 91.

Technical Abstract: GOAL: To identify the extraction method that gives the greatest yield and diversity of microbial DNA from the cecal contents of turkeys. METHODS: Eight commercially available kits and 14 different isolation protocols were evaluated. DNA was extracted as per the manufacturers' instructions from 0.2 g samples of frozen turkey cecal contents and cecal tissue. DNA quantity and quality were assessed with pico green and A260/280 measurements, and quantitative competitive PCR identified the ratio of bacterial to fungal DNA yield. Microbial diversity was estimated using Automated Ribosomal Intergenic Spacer Analysis (ARISA) based on bacterial ribosomal intergenic DNA peaks detected by an ABI3100 sequencer using the ABI GeneMapper Software v3.5. RESULTS: Agarose gel visualization of the total DNAs revealed significant DNA shearing in preparations using the Qbiogene FastDNA Kit with CLS-Y buffer, Invitrogen Easy-DNA Kit protocol #3 for small samples and the Epicentre SoilMaster DNA Extraction Kit. By pico green and A260/280 spectrophotometry, Qbiogene FastDNA Kit with CLS-Y buffer and Invitrogen Easy-DNA Kit protocol #3 for small samples yielded the greatest quantities and purity of DNA; the Invitrogen kit yielded significantly more DNA than any other method. Qbiogene FastDNA Kit with the CLS-TC buffer, QIAamp DNA Stool Mini Kit with the pathogen detection method, Roche High Pure Kit with the tissue method and MoBio Ultra Clean Fecal Kit yielded a bacterial:fungal DNA ~ 1. The MoBio Ultra Clean Soil Kit and both the tissue and bacterial methods from the Roche High Pure PCR Template Preparation Kit generated the most diversity as measured by ARISA. CONCLUSION: These results allow researchers to determine which DNA isolation method is most appropriate for their application. In studies of microbial ecology such as ours, equal isolation is essential from all members of the microbial community. For this reason, the Roche High Pure PCR Template Preparation Kit using the procedure for mammalian tissue appears to be best suited to our needs.

Last Modified: 7/30/2014
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