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United States Department of Agriculture

Agricultural Research Service

Research Project: ENHANCING SUSTAINABILITY OF FOOD PRODUCTION SYSTEMS IN THE NORTHEAST Title: Comparison of microvolume DNA quantification methods for use with volume-sensitive environmental DNA extracts

Author
item Rothrock, Michael

Submitted to: Journal of Natural and Environmental Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 13, 2011
Publication Date: August 25, 2011
Citation: Rothrock Jr, M.J. 2011. Comparison of microvolume DNA quantification methods for use with volume-sensitive environmental DNA extracts. Journal of Natural and Environmental Science. 2(1):34-38.

Interpretive Summary: Molecular-based microbial ecology studies require accurate DNA concentration estimates from environmental DNA extracts. While commercially-available kits allow for the enhanced recovery of relatively clean DNA from environmental samples, the volume of the DNA recovered (typically = 100 microliters) requires DNA concentrations to be accurately quantified using minimal volumes (= 2 microliters). Therefore, this study compared the use of absorbance-based (using the NanoDrop and Synergy H4 instruments) and fluorescence-based (using 96-well plate, or microvolume plate formats) microvolume methods to quantify DNA from a variety of different environmental and pure culture extracts. We found that absorbance-based methods overestimated the DNA concentration of environmental samples by ~50%, as compared to fluorescence-based methods using the double-stranded DNA specific dye, PicoGreen. Additionally, the use of the microvolume plate in conjunction with the PicoGreen dye, allowed for the sensitive detection of DNA from environmental samples while using 100-fold less sample and reagent volumes. This represents a significant sample volume and cost savings over the traditional PicoGreen 96-well plate method, without sacrificing sensitivity or accuracy. These results will be of particular interest to researchers who routinely characterize, quantify, and/or identify microbes and microbial communities based on their DNA extracted from agricultural and non-agricultural environments.

Technical Abstract: Accurate DNA concentration estimates from environmental samples using minimal sample volumes are essential for most downstream applications. To compare the efficacy of microvolume quantification methods, DNA was extracted from soil, compost, and pure culture samples, and quantified using two absorbance-based (NanoDrop or Synergy H4 instruments) and two fluorescence-based assays (multiwell plate or microvolume Take3 plate formats). Fluorescence-based methods using the dsDNA specific dye PicoGreen were more sensitive than the absorbance-based methods which overestimated DNA concentrations from environmental samples by ~50%, most likely due to absorbance by non-dsDNA substances in the unpurified extracts. The microvolume Take3 plate, in conjuction with PicoGreen, produced statistically similar DNA estimates to the traditional multiwell plate format for all samples, but used 100-fold less DNA sample and PicoGreen working solution volumes. This method resulted in significant sample volume and cost savings as compared to the traditional PicoGreen multiwell plate protocol, without sacrificing sensitivity or accuracy.

Last Modified: 10/25/2014