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Applied and Environmental Microbiology, October 2006, p. 6860-6862, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.01243-06

LETTER TO THE EDITOR

Possible Errors in the Interpretation of Ethidium Bromide and PicoGreen DNA Staining Results from Ethidium Monoazide-Treated DNA


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Nocker and Camper reported that DNA from dead bacterial cells (Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium) treated with ethidium monoazide (EMA) is lost during the DNA extraction procedure (2). This was concluded from the missing or less intense DNA band on an agarose gel stained with ethidium bromide in comparison to the intensities of bands in untreated samples. According to the information provided in Materials and Methods, PicoGreen-based DNA quantification of the EMA-treated samples was performed as well, albeit no data have been given. When critically reading the article, we noticed the lack of an investigation and discussion of the possible influence of EMA bound to DNA on staining the same DNA with ethidium bromide and with PicoGreen.

EMA is a monoazide derivative of ethidium bromide and has been shown to intercalate into the DNA double helix, similarly to the parent compound (5). Upon exposure to light, EMA may be covalently attached to DNA, thus blocking PCR amplification (4, 5). The rationale for using EMA for real-time PCR-based discrimination of viable and dead bacterial cells is that this chemical compound presumptively does not enter viable bacterial cells. Thus, only free DNA and DNA from dead bacteria are blocked from PCR amplification (4). Likewise, intercalation of ethidium bromide and intercalation of minor-groove binding of PicoGreen (6) might be hindered by covalently attached EMA. In addition, the fluorescence of the EMA/DNA complex might interfere with the fluorescence detection of the ethidium bromide or PicoGreen-stained DNA. It has been reported that the EMA/DNA complex has the same excitation (510-nm) and emission (600-nm) wavelengths as the ethidium bromide/DNA complex (1). However, the detection of ethidium bromide-stained DNA on agarose gels is performed under UV light.

To investigate these interactions, we performed a few simple experiments. In comparison to results with untreated DNA and depending on the DNA concentration, the detection of EMA-treated DNA on an ethidium bromide-stained agarose gel was compromised (Fig. 1). Concordantly, spectroscopic analysis of the EMA/DNA complex at an excitation wavelength representative of UV light (240 nm) using a model F-4500 fluorescence spectrophotometer (Hitachi High Technologies America, San Jose, CA) revealed no emission at wavelengths ranging from 200 to 800 nm. Two 1-ml aliquots of calf thymus DNA at a concentration of 1 ng/µl were subjected to EMA treatment and pooled for this analysis (3). In addition, a comparison of fluorescence values of untreated and EMA-treated samples gathered using the plate read mode of a model Mx3000 p real-time PCR cycler (Stratagene, La Jolla, CA) revealed that the PicoGreen DNA measurement is also compromised (Table 1).


Figure 1
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  		FIG.1. Analysis of EMA-treated and EMA-untreated DNA on an ethidium bromide-stained agarose gel. EMA treatment was performed, as published recently, in a volume of 1 ml at a concentration of 100 µg EMA (Molecular Probes, Eugene, OR)/ml (3). The samples were incubated for 5 min at 4°C in the dark, followed by exposure to light in macrocuvettes (Greiner, Frickenhausen, Germany) for 2 min at a distance of 20 cm from the light source, using a Ventilux 1250 lamp and a 650-W halogen light bulb (Hedler, Runkel, Germany). Calf thymus DNA was obtained from Sigma-Aldrich (St. Louis, MO). Salmonella enterica serovar Typhimurium NCTC 12023 DNA was isolated from an overnight culture in tryptic soy broth with 0.6% yeast at 37°C using the NucleoSpin tissue kit (Machery-Nagel, Düren, Germany). Lane m, 100-bp ladders. Lanes marked with a contain samples of DNA exposed (exp.) to light without the addition of EMA.

 

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  		TABLE 1. PicoGreen DNA measurement of untreated and EMA-treated DNA samples

 
In conclusion, the additional data presented here confirm that the missing or fainter band of the EMA/DNA complex on the ethidium bromide-stained agarose gel and the missing or reduced signal of the PicoGreen DNA measurement do not suffice to conclude that the EMA/DNA complex is actually lost during DNA isolation.


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  1. Bolton, P. H., and D. R. Kearns. 1978. Spectroscopic properties of ethidium monoazide: a fluorescent photoaffinity label for nucleic acids. Nucleic Acids Res. 5:4891-4903.[Abstract/Free Full Text]
  2. Nocker, A., and A. K. Camper. 2006. Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Appl. Environ. Microbiol. 72:1997-2004.[Abstract/Free Full Text]
  3. Nogva, H. K., S. M. Drømtørp, H. Nissen, and K. Rudi. 2003. Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR. BioTechniques 34:804-813.[Medline]
  4. Rudi, K., H. K. Nogva, B. Moen, H. Nissen, S. Bredholt, T. Møretrø, K. Naterstad, and A. Holck. 2002. Development and application of new nucleic acid-based technologies for microbial community analyses in foods. Int. J. Food Microbiol. 78:171-180.[CrossRef][Medline]
  5. Yielding, L. W., and W. J. Firth III. 1980. Structure-function characterization for ethidium photoaffinity labels as mutagens in Salmonella. Mutat. Res. 71:161-168.[Medline]
  6. Zipper, H., H. Brunner, J. Bernhagen, and F. Vitzthum. 2004. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Res. 32:e103.[Abstract/Free Full Text]
Ingeborg Hein*
Gabriele Flekna
Martin Wagner
Department of Veterinary Public Health
University of Veterinary Medicine
Veterinaerplatz 1
A-1210 Vienna, Austria

* Phone: 43 1 25077 3507, Fax: 43 1 25077 3590, E-mail: ingeborg.hein{at}vu-wien.ac.at


Authors' Reply


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Hein et al. do bring up a justified concern which we had not considered in our analysis. We confirm that covalently cross-linked ethidium monoazide (EMA) compromises DNA detection on an ethidium bromide-stained agarose gel and by PicoGreen fluorescence. PicoGreen-based DNA quantification was the basis for all diagrams in the corresponding publication (2) where relative DNA yields were shown.

We present here other evidence for our hypothesis that DNA is lost during DNA extraction: the red color of the pelleted cell debris originating from dead cells. Experiments 1 and 2 described by Nocker and Camper (2) were repeated with slight modifications to provide examples.

Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium cultures were grown for 12 h in LB broth at 37°C and diluted to an optical density at 600 nm of 1. In the first experiment, 500-µl aliquots of the cultures were either heat treated for 15 min at 72°C or exposed to isopropanol (final concentration, 70%), resulting in a complete loss of culturability. Aliquots of these killed cells and aliquots of the untreated culturable cells were subjected to EMA treatment (as described previously) or not. After being exposed to light for 1 min, cells were harvested by centrifugation for 5 min at 5,000 x g and the supernatant was carefully removed. Cell pellets were resuspended in 978 µl of sodium phosphate buffer and 122 µl MT buffer (solutions were provided by the FastDNA SPIN kit for soil; Qbiogene, Carlsbad, California). Cell lysis was achieved in lysing matrix E tubes by bead beating, using a FastPrep machine (Qbiogene) for 25 s at a speed setting of 4.5 m/s. Figure 1 shows the lysis matrix tubes with pelleted cell debris after centrifugation for 5 min at 14,000 x g. Only EMA-treated dead cells produce an intensely red color of the debris pellet, whereas the cell debris originating from EMA-treated live cells is only faintly red. The red color of the cell debris is very likely to originate from genomic DNA, as EMA has not been reported to have any significant affinity for other cell components.


Figure 1
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  		FIG.1. Lysis matrix tubes with pelleted cell debris of viable and dead Salmonella and E. coli O157:H7 without (–) or with (+) previous EMA treatment. Culture aliquots were either processed directly without stress exposure, heat treated at 72°C for 15 min, or exposed to 70% isopropanol (isoprop.) for 10 min. Cells were pelleted by centrifugation. Cell pellets were resuspended and subjected to bead beating. Lysis matrix tubes are shown after centrifugation for 5 min at 14,000 x g, with the cell debris facing upward.

 
In the second experiment, 500-µl aliquots of Salmonella were subjected to heat exposure at 72°C for increasing time periods prior to EMA treatment. Samples were processed as described above. Increasing heat exposure resulted in an increasingly red color of the cell debris pellets (Fig. 2).


Figure 2
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  		FIG. 2. Lysis matrix tubes with pelleted cell debris of Salmonella exposed to heat stress at 72°C (1 to 15 min) with subsequent EMA treatment. Cells were pelleted by centrifugation. Cell pellets were resuspended and subjected to bead beating. Lysis matrix tubes are shown after centrifugation for 5 min at 14,000 x g, with the cell debris facing upward.

 
The reason for the faint red color of debris pellets originating from viable cells has been outlined previously; EMA might not be totally membrane impermeant, correlating with the observation that EMA to some extent also decreases the DNA yield from growing, unstressed cells (results mainly with E. coli O157:H7). This problem has been solved in the meantime by substituting propidium monoazide (PMA) for EMA. PMA seems to be more membrane impermeant and does not lead to signal reduction during quantitative PCR (3). The cell debris from culturable cells treated with PMA is completely white.

Due to the fact that traditional absorbance- or fluorescence-based methods are compromised by the cross-linked dye, the final proof might be to perform DNA quantification using elemental analysis. English et al. (1) recently described a method using inductively coupled plasma-optical emission spectroscopy (ICP-OES) for that purpose, based on the stoichiometry of phosphorus within DNA. As EMA does not contain phosphorus, the DNA would be the only component contributing to a phosphorus signal. Although ICP-OES (avoiding any solutions containing phosphorus for DNA extraction) theoretically could address the question, the relatively high detection limit for phosphorus quantification (about 100 µg/ml DNA, with a required volume of several milliliters) makes measurements in the concentration range presented here unsuitable. Other alternative methods will have to be explored to provide final evidence.


   REFERENCES 
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  1. English, C. A., S. Merson, and J. T. Keer. 2006. Use of elemental analysis to determine comparative performance of established DNA quantification methods. Anal. Chem. 78:4630-4633.[Medline]
  2. Nocker, A., and A. K. Camper. 2006. Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Appl. Environ. Microbiol. 72:1997-2004.[Abstract/Free Full Text]
  3. Nocker, A., C.-Y. Cheung, and A. K. Camper. 5 June 2006, posting date. Comparison of propidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J. Microbiol. Methods [Online]. doi:10.1016/j.mimet.2006.04.015.
Andreas Nocker*
Anne K. Camper
Center for Biofilm Engineering
Montana State University
Bozeman, MT 59717-3980

* Phone: (406) 994-1849, Fax: (406) 994-6098, E-mail: anocker{at}erc.montana.edu

Applied and Environmental Microbiology, October 2006, p. 6860-6862, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.01243-06




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