Possible errors in the interpretation of ethidium bromide and PicoGreen DNA staining results from ethidium monoazide-treated DNA.

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/ l were subjected to EMA treatment and pooled for this analysis (3). In addition, a comparison of fluorescence values of untreated and EMAtreated 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). 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.