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United States Department of Agriculture

Agricultural Research Service

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

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

Submitted to: American Society for Microbiology Branch Meeting
Publication Type: Abstract Only
Publication Acceptance Date: October 29, 2004
Publication Date: November 12, 2004
Citation: Scupham, A.J., Wesley, I.V. 2004. Extraction of total microbial DNA from the cecal contents of turkeys: comparison of methods [abstract]. North Central Branch of the American Society for Microbiology. Paper No. 67.

Technical Abstract: GOAL: To compare turkey cecal microbial diversity present in DNA samples from eight commercially available DNA isolation kits. METHODS: 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 v. 3.5. 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 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 MoBio Ultra Clean Soil DNA Isolation Kit and the Epicentre Soilmaster DNA Extraction Kit yielded the most bacterial 16S DNA copies/ng total DNA. The Epicentre Soilmaster DNA Extraction Kit and Qbiogene FastDNA Kit with CLS-TC and BoomL6 buffers generated the greatest diversity as measured by ARISA. CONCLUSION: These results indicate bacterial diversity generated by a DNA isolation method is not necessarily correlated to the quantity of DNA isolated by that method and allows researchers to determine which DNA isolation method is most appropriate for their applications. Studies of microbial ecology must accurately reflect the microbial community in question, therefore it is essential to isolate equivalent amounts of DNA from all members of the microbial community. For this reason, the Qbiogene FastDNA Kit with CLS-TC buffer appears to be best suited to our needs.

Last Modified: 8/21/2014
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