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United States Department of Agriculture

Agricultural Research Service

Title: Importance of DNA Isolation Method on Representation of Microbial Diversity

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

Submitted to: Proceedings of North Central Avian Disease Conference
Publication Type: Abstract Only
Publication Acceptance Date: October 3, 2004
Publication Date: October 3, 2004
Citation: Scupham, A.J., Rardin, J.A., Wesley, I.V. 2004. Importance of DNA isolation method on representation of microbial diversity [abstract]. In: Proceedings of the North Central Avian Disease Conference. The Food Safety Consortium Annual Meeting, October 3-5, 2004, Ames, Iowa. 2004 CDROM.

Technical Abstract: To compare turkey cecal microbial diversity present in DNA samples from eight commercially available DNA isolation kits. Fourteen different isolation protocols from eight commercially available kits 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 competitive PCR quantified the bacterial component. 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. 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 DNA of the greatest purity; the Invitrogen Easy-DNA Kit protocol #4 for large samples yielded significantly more DNA than any other method. Competitive PCR showed the MoBioSoil Kit yielded the most bacterial 16S DNA copies/pg total DNA. The Roche High Pure PCR Template Preparation Kit, bacterial method and Qbiogene FastDNA Kit with BoomL6 buffer generated the greatest diversity as measured by ARISA. These results indicate bacterial diversity generated by a DNA isolation method is not necessarily correlated to amount of DNA isolated by that method and allows 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: 12/19/2014
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