Genomics Improve Sensitivity and Performance with Non-Mutagenic DNA-Binding Dyes

Biotium, Inc. is a biotechnology company dedicated to providing the best innovative reagents for life science research. We focus on novel fluorescent products for genomics, proteomics and cellular analyses. Some of our most popular products include nucleic acid binding dyes for qPCR and electrophoresis, protein gel stains, and reactive dyes for protein labeling.

GELRED AND GELGREEN NUCLEIC ACID GEL STAINS GelRed™ and GelGreen™ dyes are a new generation of fluorescent nucleic acid gel stains designed to replace the highly toxic (EtBr). Developed by scientists at Biotium, GelRed and GelGreen are superior to EtBr and other EtBr alternatives by having a combination of low toxicity, high sensitivity, and exceptional stability. Figure 2A Figure 2B For decades, EtBr has been the predominant dye used for nucleic Figure 2: Comparison of GelRed and ethidium bromide (EtBr) acid gel because of its low price and generally sufficient in precast (A) and post gel staining (B) using 1% gels in sensitivity. However, EtBr is a highly mutagenic compound; the TBE buffer. Two-fold dilutions of ladder were loaded onto each gel hazards and costs associated with EtBr decontamination and waste in 4 lanes in the amounts of 200, 100, 50 and 25 ng, respectively, disposal can ultimately make the dye expensive to use. For this from left to right. Gels were imaged using a UV transilluminator reason, alternative gel stains, such as SYBR® dyes, have become and photographed. commercially available in recent years. Although these alternative dyes have reduced mutagenicity, they often have to sacrifice other than EtBr. SYBR dyes also enter cells rapidly to stain mitochondria aspects of the dyes. For example, SYBR Safe has very limited and nuclear DNA, making it more likely for the dyes to be toxic sensitivity while SYBR Green and SYBR Gold are much less stable at high enough concentrations. Also, SYBR Green I is known to strongly potentiate mutation caused by UV light or another (Ohta, et al. Mutat. Res. 492, 91(2001)).

GelGreen GelGreen GelRed and GelGreen dyes are highly sensitive either as precast Excitation Emission or post gel stains. Designed primarily for use with a 302 nm UV GelRed GelRed Excitation Emission transilluminator (Figure 1), GelRed is much more sensitive than EtBr. In precast gel staining, it is well known that EtBr migrates in the opposite direction of DNA, resulting in high background in the high molecular weight DNA region and limited sensitivity with small DNA fragments (Figure 2A). However, GelRed does not migrate and diffuse out of the gel as easily as EtBr, resulting in more even staining of DNA fragments. Moreover, post-staining with EtBr results in high background and destaining is often necessary to remove excess dye (Figure 2B). Unlike EtBr, destaining is not necessary with GelRed 200 300 400 500 600 700 as it has low intrinsic fluorescence and only becomes highly Wavelength (nm) fluorescent upon binding to nucleic acids. GelGreen was developed Figure 1: Normalized excitation and emission spectra of GelRed to meet the needs of researchers who use a 488 nm laser-based (red) and GelGreen (green) in the presence of dsDNA. gel scanner or a system that uses a visible blue light for excitation. GelGreen is spectrally similar to SYBR Safe, but is far more sensitive (Figure 3).

16 Life Science Magazine • vwr.com • 1.800.932.5000 Genomics

GelGreen SYBR® Safe SYBR Safe GelRed GelGreen Bright-field Fluorescence

Figure 3: Comparison of GelGreen and SYBR Figure 4: HeLa cells were incubated with 1X SYBR Safe, GelRed or GelGreen. Images Safe in precast gel staining using 1% agarose were taken following incubation for 30 min. SYBR Safe readily entered cells as gel in TBE buffer. Two fold serial dilutions of evident from the bright green nuclear staining. However, no fluorescence staining ladder were loaded onto each gel in 4 lanes is evident in GelRed or GelGreen stained cells. in the amounts of 200, 100, 50 and 25 ng, respectively, from left to right. Gels were imaged using a 302 nm transilluminator and photographed with a SYBR filter using the GelDoc-It ® imaging system.

To make GelRed and GelGreen safe, scientists at Biotium used a novel yet very simple concept: reduce genotoxicity by preventing the dyes from entering live cells. A DNA-binding dye can be made non-mutagenic by denying its chance to be in contact with genomic DNA in live cells. The chemical structures of GelRed and GelGreen dyes were engineered such that the dyes are incapable of crossing cell membranes (Figure 4). Moreover, the Ames test confirmed that GelRed and GelGreen are non-mutagenic and noncytotoxic at concentrations well above their working concentrations (Figure 5). Furthermore, environmental safety tests showed that GelRed Figure 5: Comparison of mutagenicity among GelRed, GelGreen and ethidium and GelGreen are non-hazardous and nontoxic bromide (EB). Ames tests were performed in Salmonella strain TA98 with S9 to aquatic life. GelRed and GelGreen successfully metabolic activation. passed the Aquatic Toxicity Test (CCR Title 22) * indicates EB not tested at these concentrations. based on the EPA/600/4-85/013 protocol. As a result, GelRed and GelGreen can be disposed of down the drain or with regular trash. For more information, please contact your VWR Life Science Specialist.

Life Science Magazine • vwr.com • 1.800.932.5000 17 Genomics

4 Other major advantages of GelRed and GelGreen are their Fast Eva Green Master Mix from Biotium remarkable sensitivity, long shelf-life, and photostability. Recently, SYBR Green Master Mix from Company A Biotium has developed a prestaining kit containing optimally SYBR Green Master Mix from Company Q formulated GelRed or GelGreen loading buffer and a superior 3 running buffer that results in sharp resolution of DNA bands and is amenable to high-voltage electrophoresis to shorten running time. 2

This new method allows DNA samples to be loaded directly with AFU GelRed or GelGreen and visualized after electrophoresis (Figure 6).

3 Staining Protocols 1

GelRed Visualize gel

0 Precast Cast gel 15 20 25 30 35 40 and load Cycle Number agarose DNA Fast EvaGreen Master Mix from Biotium SYBR Green Master Mix from Company A SYBR Green Master Mix from Company Q Incubate gel Cast gel in GelRed Post-stain and load Comparison among Fast EvaGreen master mix from solution Figure 7A: DNA Biotium and two fast SYBR Green master mixes from Companies A and Q in the amplification of a APTG fragment of human genomic DNA. Reactions were carried out on the same plate using DNA + ABI 7900 Fast on ⌬NA inputs of 0-200 ng. The passive reference Prestaining GelRed or Cast gel GelGreen and load dye ROX was adjusted for each master mix so that the final DRn loading buffer DNA faithfully reflects the fluorescence change of each master mix.

Figure 6: Three methods for GelRed and GelGreen staining.

EVAGREEN® DYE EvaGreen dye is a nucleic acid binding dye, which has been shown to be less mutagenic than SYBR® Green. Our EvaGreen dye is ideal for both real-time or quantitative PCR (qPCR) and melt curve analysis. The dye selectively binds to dsDNA via a novel “release-on- demand” mechanism that ensures low PCR inhibition and permits melt curve analysis at below saturation dye concentrations. The EvaGreen master mixes have been tested on a variety of templates. In this example (Figure 7A), the results show that the Fast EvaGreen master mix is significantly more sensitive and gives a wider linear detection range than master mixes with SYBR Green dye from other companies. More specifically, the EvaGreen master mix gives early Ct values and a wide 5-log linear detection range compared to master mixes from Companies Q and A. On the other hand, the master mix from Company A in particular suffered from significant Ct delay and narrow linear detection range as suggested by the Figure 7B: Comparison of Fast EvaGreen master mix and fast irregular spacing between the curves. In addition to its qPCR SYBR Green master mixes from Companies A and Q in melt curve performance, EvaGreen dye can also be used in melt curve analyses. analysis. Analyses were carried out following the amplification Figure 7B illustrates the superior sensitivity and sharp melt peak of of 200 ng of human genomic DNA using each of the three the product generated from the Fast EvaGreen master mix compared master mixes. with fast SYBR Green master mixes from Companies Q and A.

18 Life Science Magazine • vwr.com • 1.800.932.5000 Genomics

PROPIDIUM MONOAZIDE (PMA™) cell membrane-impermeable; therefore, only dead bacteria are susceptible to DNA modification due to compromised cell The ability to selectively detect viable bacteria in the presence of membranes. Thus, pre-treating a sample with PMA prior to lysis dead bacteria with high sensitivity is of vital importance for many of bacteria and subsequent PCR analysis permits the detection practical applications, including safety inspection of food products, of only viable bacteria in a highly sensitive and reliable manner. drinking water quality control, and medical diagnosis. The traditional detection method of bacterial culturing is time-consuming and lacks In general, the difference in crossing threshold (Ct) values of sensitivity. Detection based on PCR is a rapid and highly sensitive live cells compared to the corresponding sample without PMA alternative method; however, PCR methods cannot distinguish live treatment was 1-2 Ct or less (Figure 8B). Heat-killed samples from dead cells, making interpretation of analytical results difficult. demonstrated a difference of about 12 Ct when compared to The novel DNA-modifying dye PMA developed by Biotium overcomes cells without PMA. These results suggest that PMA can be used this problem. in conjunction with PCR to distinguish between live and dead bacteria, and is also effective in mixed populations of live and PMA is a photo-reactive dye with a high affinity for DNA. The dead cells (data not shown). dye intercalates into dsDNA and forms a covalent linkage upon exposure to intense visible light. The chemically-modified DNA cannot be amplified by PCR (Figure 8A). PMA is designed to be Description Unit Size Cat. No. Price Cheetah™ Hot Start Taq polymerase 500U 89138-878 210.00 EvaGreen, 20X in Water 5 x 1mL 89138-982 157.50 GelRed 10,000X in Water 0.5mL 89139-138 100.00 GelGreen 10,000X in Water 0.5mL 89139-146 95.00 Fast Probe Master Mix 200 x 20µL rxn 89139-002 150.00 Fast Probe Master Mix - ROX 200 x 20µL rxn 89139-042 150.00 PMA™ in water, 20 mM 100µL 89139-068 110.00 PMA dye 1mg 89139-058 105.00 Fast EvaGreen Master Mixes for qPCR and HRM No ROX 200 x 20µL rxn 89138-990 126.00 Low ROX 200 x 20µL rxn 89139-026 126.00 Figure 8A: Principle of the technology. High ROX 200 x 20µL rxn 89139-034 125.00

Figure 8B: Representative quantitation analysis of real-time PCR performed on live and dead E. coli treated with and without PMA using primers against a region of the 16S rRNA.

Life Science Magazine • vwr.com • 1.800.932.5000 19