Low-cost, ultra-sensitive fluorescence detection of DNA by gel electrophoresis using environmentally benign Gelite™ Safe DNA Gel Stain
Gelite™ Safe DNA Gel Stain has been developed specifically to be a less hazardous alternative to ethidium bromide (EtBr) and SYBR® Green dyes for staining DNA in agarose and polyacrylamide gels, without sacrificing detection sensitivity. The exceptional sensitivity and strong DNA binding affinity of Gelite™ Safe DNA Gel Stain allows for DNA to be stained without any requirement for destaining. In addition to its superior binding properties, Gelite™ Safe DNA Gel Stain is essentially non-fluorescent in the absence of nucleic acids, displaying very low gel matrix background fluorescence. Upon binding to nucleic acids, Gelite™ Safe DNA Gel Stain exhibits a significant fluorescence enhancement, several orders of magnitude greater than that of EtBr. Gelite™ Safe DNA Gel Stain was optimized to be compatible with various gel documentation and imaging instruments, including UV and visible light transilluminators, CCD-camera-based gel documentation systems, and laser scanners. It is the first single formulation that can be imaged either in the green or red emission channel of such devices. Unlike the membrane-permeant SYBR® Green dye, which is highly toxic to cells and the environment, the membrane-impermeant properties of Gelite™ Safe DNA Gel Stain make it a much safer, noncytotoxic alternative, as demonstrated in the WST-8 cytotoxicity assay. Furthermore, Ames testing has confirmed Gelite™ Safe DNA Gel Stain is significantly less mutagenic than EtBr and SYBR® Green stains, even at concentrations well above the working concentration required for gel staining.
Ethidium bromide (EtBr) is an intercalating agent commonly employed as a fluorescent nucleic acid stain in biomedical research laboratories for analytical techniques such as agarose gel electrophoresis (1, 2). The health hazards of working with toxic chemicals, such as EtBr, are well recognized, as are the accompanying economic considerations of steeply rising costs that accompany appropriate handling and disposal of mixed waste (1, 2). Conventional DNA staining procedures in agarose and polyacrylamide gels generate significant amounts of toxic waste, creating a need for better reagent alternatives that minimize environmental impact. Furthermore, EtBr is typically visualized using a high energy, 312 nm UV –transilluminator, which itself can represent a hazard to laboratory personnel, potentially leading to sunburn, photokeratitis, photoconjunctivitis, retinal burns, cataracts, and even blindness, if appropriate operating precautions are not heeded (3).
The cyanine dye, Gelite™ Safe DNA Gel Stain, represents the newest addition to AAT Bioquest's growing family of nucleic acid detection reagents. Gelite™ Safe DNA Gel Stain, provides a greatly improved safety profile and uncompromised detection sensitivity, serving as an environmentally benign alternative to conventional DNA stains for gel electrophoresis. Upon binding to DNA, the resulting DNA-dye-complex absorbs visible green light with ?max of 512 nm, which is readily detected using Blue or Blue-Green LED systems, but also absorbs significantly in the UV region of the spectrum and is thus readily detectable with a UV light source. The DNA-dye complexes emit in the yellow region of the visible light spectrum, with ?max centered at about 550 nm (Figure 1). The emission peak is relatively broad, facilitating detection of the dye in the green through red regions of the visible light spectrum, thus ensuring compatibility with a wide range of gel documentation and imaging systems.
Normalized excitation (left) and emission (right) spectra for Gelite™ Safe DNA Gel Stain bound to DNA in TE buffer. DNA (Calf DNA stock solution: 0.85 mg/mL Sigma D1501) was added at a final concentration of 3 µg/mL. Excitation-emission spectra were measured after addition of the DNA, using a Varian Cary Eclipse fluorescence spectrophotometer (Agilent, Santa Clara, CA).
Human and Environmental Safety
AAT Bioquest is committed to designing our products to be safe to use and environmentally friendly. We endeavor to enable our customers to make the world healthier, cleaner, and safer. Ethidium bromide (EtBr) has been commonly used as a DNA stain for decades. However, EtBr is harmful if ingested and is quite toxic if inhaled. EtBr has been shown to be mutagenic in various tests and is also an aquatic toxin. SYBR® Safe stain was introduced as a safer alternative to EtBr and SYBR® Green dye, but unfortunately, it is much less sensitive than SYBR® Green stain, only displaying sensitivity comparable to EtBr (4).
Gelite™ Safe DNA Gel Stain is a safer alternative to conventional dyes, such as ethidium bromide and SYBR® Green I dye (5, 6). This is readily demonstrated using widely employed in vitro biological assays. Upon testing with the Ames mutagenicity or the WST-8 cytotoxicity assays, Gelite™ Safe DNA Gel Stain exhibits very low mutagenicity and low cytotoxicity (Figure 2). This contrasts markedly with other commonly implemented gel stains such as EtBr, SYBR® Green I, SYBR® Gold, and GelRed® stains (4). Additionally, the DNA stain is stable at room temperature, facilitating long-term storage without consuming valuable refrigeration or freezer space. The non-toxic, environmentally friendly nature of the dye simplifies its disposal, which can be accomplished by discarding into the regular trash.
Representative DNA gel stain mutagenicity and cytotoxicity profiles. (A.) Mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test). (B.) Cytotoxicity in the WST-8 Cytotoxicity Assay. WST-8 is bio-reduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells.
Detection Sensitivity and Instrument Compatibility
With Gelite™ Safe DNA Gel Stain, there is no need to sacrifice DNA detection sensitivity for safety (Figure 3). While the limit of DNA detection for SYBR® Safe gel stain is approximately 3 ng, Gelite™ Safe DNA Gel Stain detects as little as 0.67 ng DNA. Additionally, the gel stain provides greater sensitivity, with lower background fluorescence, than most conventional DNA gel stains (4).
Gelite™ Safe DNA Gel Stain does not readily penetrate eukaryotic cells. HeLa cells were incubated for 30 minutes at 37°C with 1X DNA gel stain. EtBr, SYBR® Green I and SYBR® Safe dyes rapidly bound to DNA in living cells, resulting in bright nuclear staining. Gelite™ Safe DNA Gel Stain failed to penetrate living cells and thus was unable to bind to their DNA, as shown by an absence of fluorescence.
The dye is compatible with denaturing and native agarose and polyacrylamide gels. It can be imaged with various standard imaging systems, such as a 360 nm black light UV epi- or trans-illuminator with a film or digital camera, laser scanner, or Blue-Green LED (490–495 nm) digital gel documentation systems. Gelite™ Safe dye-stained DNA can readily be visualized using imaging systems equipped with lasers that emit at 473, 488, or 532 nm. Instrument manufacturers can be consulted for optimal filter settings to use in combination with Gelite™ Safe DNA Gel Stain. One important feature of Gelite™ Safe DNA Gel Stain is that the dye maximally absorbs at around 500 nm, allowing it to be excited by visible LED light sources. Visible light illumination avoids personnel exposure to harmful UV irradiation. In addition, cloning efficiency with gel-purified DNA is improved with visible light illuminators, likely because they avoid UV damage to DNA during gel excision (7). Generally speaking, Gelite™ Safe DNA Gel Stain has an excitation/emission profile similar to SYBR® Gold or SYBR® Safe dye and can be imaged and documented with any system that provides filters or settings optimized for these dyes (8).
DNA separation and detection by agarose gel electrophoresis is considered one of the most frequently employed techniques in biomedical research laboratories. Traditionally, DNA fragments loaded on agarose gels are stained with ethidium bromide and detected using a UV-transilluminator-based system. Gelite™ Safe DNA Gel Stain is an ultra-sensitive and low-cost dye suitable for DNA detection after denaturing or nondenaturing agarose or polyacrylamide gel electrophoresis. The dye can be used by prior incorporation into the matrix of precast gels or post-electrophoresis gel staining. Loading of the dye in sample wells is not recommended, however, since tight binding of the dye with the DNA can lead to migration anomalies during the electrophoretic separation. This phenomenon also occurs with other commonly used DNA gel stains, such as SYBR® Green and SYBR® Gold stains. Following staining, DNA can be extracted from the gel using either a column-based PCR clean-up system or an ethanol precipitation procedure. Greater than 99% of the stain can be removed from double-stranded DNA simply by ethanol precipitation.
Comparison of DNA detection in 1% agarose gel in TBE buffer using Gelite™ Safe, EtBr, and SYBR® Safe. Two-fold serial dilutions of 1 kb DNA ladder were loaded in amounts of 100 ng, 50 ng, and 25 ng from left to right. Gels were stained for 60 minutes with Gelite™ Safe, EtBr, and SYBR® Safe according to the manufacturer's recommended concentrations and imaged using the ChemiDoc™ Imaging System (Bio-Rad®). Gels were illuminated using a 300 nm transilluminator fitted with GelGreen and GelRed filters.
DNA is typically detected in agarose gels using fluorescent dyes, such as EtBr, SYBR® Green, or SYBR® Safe stains, or by silver staining procedures (3, 9). The advantages of Gelite™ Safe DNA Gel Stain compared with other commercially available DNA staining technologies has been shown to be high sensitivity detection, combined with low toxicity. Additionally, compared with silver staining, the Gelite™ Safe DNA Gel Stain is environmentally friendly and less of a nuisance to laboratory personnel due to the absence of noxious solvent fumes. Unlike some silver staining methods, heavy metal ions, sodium hydroxide, ammonia, and formaldehyde are not required when using the fluorescent stain (9). Gelite™ Safe DNA Gel Stain allows sensitive detection of DNA using gentle staining conditions and harmless visible light sources that minimize artefactual damage to nucleic acids. Thus, the gentle staining procedure for DNA detection using Gelite™ Safe DNA Gel Stain should have benefits with respect to the elution of DNA from polyacrylamide and agarose gels for cloning and sequencing applications. Staining gels with Gelite™ Safe dye should be particularly attractive in educational settings, especially in combination with a harmless visible light illumination source, such as a Dark Reader? (Clare Research Chemical, Boulder, CO) or FastGene Blue/Green LED Gel Transilluminator (Bulldog Bio, Portsmouth, NH).
Table 1. Product ordering information for Gelite™ Safe DNA Gel Stain
|Gelite™ Safe DNA Gel Stain *10,000X Water Solution*||100 µL||17700|
|Gelite™ Safe DNA Gel Stain *10,000X Water Solution*||500 µL||17701|
|Gelite™ Safe DNA Gel Stain *10,000X Water Solution*||1 mL||17702|
|Gelite™ Safe DNA Gel Stain *10,000X Water Solution*||10 mL||17703|
|Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*||100 µL||17704|
|Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*||500 µL||17705|
|Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*||1 mL||17706|
|Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*||10 mL||17707|
|Gelite™ Safe DNA Gel Stain *GelRed Replacement, 10,000X in water*||0.5 mL||17708|
|Gelite™ Safe DNA Gel Stain *GelRed Replacement, 10,000X in DMSO*||0.5 mL||17709|
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- Singer VL, Lawlor TE, Yue S. 'Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test).' Mutat Res. 1999 Feb 2; 439 (1):37-47.
- Ohta T, Tokishita S, Yamagata H. 'Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli.'Mutat Res. 2001 May 31; 492 (1-2):91-7.
- Gründemann, D, Schömig, E. 'Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light.' BioTechniques 1996, 21 (5), 898.
- Tuma RS, Beaudet MP, Jin X, Jones LJ, Cheung CY, Yue S, Singer VL. 'Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators.' Anal Biochem. 1999 Mar 15; 268 (2): 278-88.
- Liu W, Li R, Ayalew H, Xia Y, Bai G, Yan G, Siddique KHM, Guo P. 'Development of a simple and effective silver staining protocol for detection of DNA fragments.' Electrophoresis. 2017 Apr;38 (8):1175-1178.