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PLoS One. 2012;7(3):e33865. doi: 10.1371/journal.pone.0033865. Epub 2012 Mar 23.

Evaluation of methods for the extraction and purification of DNA from the human microbiome.

Author information

1
Department of Biological Sciences, University of Idaho, Moscow, Idaho, United States of America.

Abstract

BACKGROUND:

DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled.

METHODOLOGY/PRINCIPAL FINDINGS:

In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used.

CONCLUSIONS/SIGNIFICANCE:

Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.

PMID:
22457796
PMCID:
PMC3311548
DOI:
10.1371/journal.pone.0033865
[Indexed for MEDLINE]
Free PMC Article

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