|Thurston Enriquez, Jeanette|
Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: September 1, 2005
Publication Date: January 1, 2006
Citation: Johnson, D.M., Thurston Enriquez, J.A. 2006. Comparison of dna extraction methods on dairy constructed wetland wastewater. American Society for Microbiology 106th General Meeting in Orlando Florida. May 21-25th, 2006. American Society for Microbiology. Interpretive Summary: Microbial analysis of environmental samples such as wastewater and soil is challenging to researchers due to the poor recovery (low concentrations) of current methods. Furthermore, differences between methods have been shown to affect the types of microorganisms detected within the sample. The current study assessed two methods for their ability to recover microbial DNA. We also determined if there were any differences in the types of microorganisms detected by each method (microbial diversity). Wastewater samples were collected from a constructed wetland system designed to treat dairy cattle wastewater. These samples underwent two different DNA extraction methods. The concentration of DNA and the microbial diversity within each sample was analyzed by both methods. Side-by-side comparisons of the two methods revealed that the MoBio method extracted significantly more DNA over the Qiagen method. The types of microorganisms recovered by both methods (microbial diversity), however, were similar between the two methods.
Technical Abstract: Direct DNA extraction from environmental samples is a useful and culture-independent method for the examination of microbial diversity. To date, there is little information on the effectiveness of commercial DNA extraction kits on wastewater. We compared two commercial DNA extraction kits for amount of DNA yielded and bacterial diversity of sequences isolated from three different dairy cattle wetland wastewater samples. DNA extraction efficiency was significantly higher (p<0.0001) using the the bead beating method (Mo Bio, UltraCleanTM Soil DNA Kit) compared to the heat lysis method (Qiagen DNA Stool Mini Kit), averaging 567.5 ng/ml (std. dev.: 430.2) and 40.2 ng/ml (std. dev.: 44.3) DNA, respectively. Neither kit extracted significantly different DNA concentrations between wetland sites (MoBio: p=0.835, Qiagen: p=0.562). The effect of sample freezing on DNA recovery efficiency prior to DNA extraction was also examined. DNA concentrations of extraction solutions from samples extracted within 24 hr of sampling were measured and compared to extractions from the same samples after they had been frozen overnight. The Qiagen kit extractions did not differ significantly (p=0.849) between fresh and frozen samples. MoBio extractions decreased significantly (p<0.0001) after freezing, from 100.9 ng/ml to 22.8 ng/ml. To determine if sequence diversity is affected by the two DNA extraction methods, PCR-DGGE analysis of DNA extraction solutions was performed for several samples. Results indicate little observable differences between the kits since an equal number of gel bands was observed in comparative studies.