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EvaGreen® & EvaGreen® Plus: Next-Generation qPCR Dyes
EvaGreen® Dye is a next-generation DNA-binding dye that is ideal for quantitative real-time PCR (qPCR) and many other applications. Biotium scientists designed the dye by taking into consideration several essential dye properties relevant to PCR, including PCR inhibition, safety, stability and fluorescence spectra of the dye. The result is a dye superior to SYBR® Green I and other commercial PCR or high-resolution melt curve (HRM) dyes.*
EvaGreen® Plus Dye is an improved alternative to Biotium’s original EvaGreen® Dye which retains the same essential benefits while providing a greater signal-to-noise ratio. The lower background and higher signal from EvaGreen® Plus provides further advantages for digital PCR and isothermal applications.
EvaGreen® Dye is used in all of Biotium’s dye-based qPCR master mixes, which combine superior brightness and sensitivity, with the ability to do sensitive melt curve analysis in the same reaction.
Visit the Forget-Me-Not™ EvaGreen® qPCR Master Mix technology page to learn more about the advantages of our qPCR reagents.
Mechanism of DNA Binding
EvaGreen® DNA-binding dye uses a novel "release-on-demand" mechanism. The dye consists of two monomeric DNA-binding units linked by a flexible spacer. Without DNA, it forms an inactive looped conformation. When DNA is present, it shifts to a conformation that binds DNA and emits fluorescence. This equilibrium continuously supplies active dye as more DNA forms during PCR.
Superior qPCR Performance
The chemical equilibrium of the looped conformation and the active form of the dye provides a unique mechanism to continuously supply the active form of the dye from the “reserve”. Consequently, an EvaGreen® master mix can be formulated with a relatively high dye concentration to maximize fluorescence signal without PCR inhibition, making the mix suitable for both qPCR and HRM applications.*
Advantages of EvaGreen® qPCR Dye
- Superior for qPCR and isothermal amplification: Brighter than SYBR® Green I due to novel “release-on-demand” DNA-binding mechanism.
- Unrivaled high resolution melt curve performance: Low PCR inhibition permits saturating dye concentration for maximal signal.
- Multiplex PCR compatible: No dye migration between amplicons allows detection of multiple products via melt curves.
Features
- Environmentally safe
Non-mutagenic, non-cytotoxic and safe to aquatic life allowing safe handling and easy disposal.
- Extremely stable
Stable during storage and PCR conditions.
- Dual purpose
Functions as both qPCR dye and DNA gel stain. Visualize PCR products directly on gel using UV or blue light without additional staining.
- Versatile applications
The only qPCR dye used in droplet digital PCR (ddPCR). Also works for isothermal amplification, microfluidic PCR systems, and capillary gel electrophoresis.
A PCR dye emits fluorescence by forming dye-DNA complexes, but this interaction inevitably causes some PCR interference. Inhibition is particularly significant early in PCR when the dye-to-DNA ratio is high. However, sufficient dye concentration remains crucial for a good signal. Therefore, optimal dye concentration must be used to achieve reliable PCR performance. EvaGreen® Dye is less inhibitory than SYBR® green, allowing for higher concentrations and much brighter signal.
Superior Melt Curve Performance
For many DNA-binding PCR dyes like SYBR® Green I, optimal concentration is low, limiting PCR signal and making them unsuitable for high-resolution melt curve analysis. Additionally, master mixes with low SYBR® Green concentration may fail to detect multiple amplicons by melt peaks due to dye migration from small to large amplicons, falsely showing a single clean product when several may actually be present. EvaGreen® Dye has a release on demand mechanism which allows the master mix to be formulated with relative high dye concentration and maximize fluorescence signal without PCR inhibition for improved HRM results.
EvaGreen® Dye
| Product Name | Catalog Number | Size |
|---|---|---|
| EvaGreen® Dye, 20X in Water (trial size) | 31000-T | 1 mL |
| EvaGreen® Dye, 20X in Water | 31000 | 5 x 1 mL |
| EvaGreen® Dye, 2000X in DMSO | 31019 | 50 uL |
| EvaGreen® Plus Dye, 20X in Water (trial size) | 31077-T | 1 mL |
| EvaGreen® Plus Dye, 20X in Water | 31077 | 5 x 1 mL |
The Industry Standard For Droplet Digital PCR
See the full list of references here.
Highlighted Citation
ddPCR technology has enabled quantitative multiplex PCR by partitioning samples into thousands of droplets that can be individually measured. Initially designed for fluorescent probe-based detection, McDermott et al. demonstrate how EvaGreen® Dye can be used with ddPCR to analyze multiple targets within a sample by distinguishing the amplified DNA mass per droplet. Results show EvaGreen®-based ddPCR performs comparably with TaqMan®-based ddPCR.
Evagreen® Dye Is Validated For Droplet Digital PCR (ddPCR)
By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction.
EvaGreen® Dye Safety
Another major advantage of EvaGreen® Dye over other PCR and HRM dyes is its safety. EvaGreen® Dye is the first and only PCR dye to date designed to be environmentally safe.
Very few PCR dyes have been thoroughly studied for their safety despite the increasing use of PCR in research and diagnostics and the fact that DNA-binding dyes are inherently dangerous due to their potential to cause mutation.
Handling and disposal of PCR master mixes raises health and environmental concerns. SYBR® Green I is more toxic than ethidium bromide and may interfere with DNA repair, increasing genotoxicity and UV-induced DNA damage. Other PCR and HRM dyes (SYTO9, LC Green, BRYT Green, ResoLight) also enter cells quickly, posing potential genotoxic risks, though safety data are not available.
EvaGreen® Dye is Membrane Impermeant
Biotium’s scientists made EvaGreen® Dye safer by increasing its size and charge, preventing cell entry and genomic DNA interaction. Independent tests confirmed it is non-mutagenic, non-cytotoxic, and safe for aquatic life. EvaGreen® Dye meets California (CCR title 22) environmental waste rules, allowing easy drain disposal. See the EvaGreen® Safety Report for details.
EvaGreen® Dye Stability
EvaGreen® Dye is very stable both during storage and under PCR conditions. EvaGreen® Dye has been reported to be stable at temperatures as high as 65°C for up to 6 months of use in the field for real-time PCR (Mil Med. 2014 179(6):626-32).
SYBR® Green I, on the other hand, is known to degrade following multiple freeze-thaw cycles and under PCR conditions. Moreover, decomposed SYBR® Green I is reported to be even more inhibitory to PCR. Thus, when assessing the performance of an EvaGreen®-based master mix, you can eliminate the stability of the dye as a variable.
Other EvaGreen® Dye Applications
EvaGreen® Dye has been applied in numerous other applications, such as Fluidigm® microfluidic PCR systems, isothermal amplification, capillary gel electrophoresis, DNA quantitation in solution and selective detection of dead cells in cell viability tests. Biotium offers EvaGreen® stand-alone dye, EvaGreen® qPCR master mixes, and other reagents and kits for qPCR.
