EVAGREEN® DYE

EvaGreen® dye is a next-generation DNA-binding dye with features ideal for use in 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 of our efforts is a dye superior to SYBR® Green I and other commercial PCR or high-resolution melt curve (HRM) dyes.*

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.

In addition, EvaGreen® dye is non-toxic, non-mutagenic, and not hazardous to aquatic life.

EvaGreen® DNA-binding dye is designed using a novel concept of DNA binding via “release-on-demand” mechanism (Fig. 1). The dye is constructed of two monomeric DNA-binding dyes linked by a flexible spacer. In the absence of DNA, the dimeric dye assumes a looped conformation that is inactive in DNA binding. When DNA is available, the looped conformation shifts via an equilibrium to a random conformation that is capable of binding to DNA to emit fluorescence. This chemical equilibrium provides a

• Environmentally safe
  Non-mutagenic, non-cytotoxic and safe to aquatic life for safe handling and easy disposal down the drain.
• Superior for qPCR and isothermal amplification
  Far brighter than SYBR® Green I for detecting amplification due to its novel “release-on-demand” DNA-binding mechanism.
• Unrivaled DNA melt curve performance
  Low PCR inhibition permits the use of saturating dye concentration for maximal signal and high-resolution DNA melt analysis.
• Extremely stable
  Stable during storage or under PCR conditions.

Superior PCR Performance

A PCR dye emits fluorescence by forming a dye-DNA complex. The interaction with DNA inevitably leads to some PCR interference. PCR inhibition by the dye can be particularly serious at the early stage of PCR, where the dye-to-DNA ratio is high. On the other hand, a sufficient dye concentration is important for generating good signal. Thus, an optimal dye concentration must be used in order to attain reliable PCR performance.

For many current DNA-binding PCR dyes, such as SYBR® Green I, the optimal dye concentration can be quite low, which limits PCR signal and also makes the dyes unsuitable for high-resolution melt curve (HRM) analysis. Furthermore, a master mix with low SYBR® Green concentration may fail to detect multiple amplicons by melt peaks due to dye migration from small amplicons to large amplicons, giving the false result of a clean single amplicon for a PCR that may, in fact, produce several products.

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.

Thus, handling and disposal of PCR master mixes can be a health and environmental issue. Indeed, SYBR® Green I is found to be even more environmentally toxic than ethidium bromide, one of the best known mutagens. SYBR® Green I has been suggested to interfere with DNA repair mechanisms in cells, and as a result it potentiates genotoxicity of chemicals as well as DNA damage by UV light.

Although no safety data are available on other PCR and HRM dyes (e.g., SYTO9, LC Green, BRYT Green and ResoLight), those dyes are all known to enter cells in a matter of minutes, thus posing potential genotoxicity risk. With this in mind, Biotium’s scientists designed EvaGreen® dye to be cell membrane impermeable by increasing the molecular size and charge of the dye (Figure 4). Because EvaGreen® dye is denied the chance to interact with genomic DNA in living cells, it is made much safer than the other dyes. Independent laboratory tests have confirmed that EvaGreen® is nonmutagenic, noncytotoxic and safe to aquatic life. The dye has passed environmental hazardous waste regulation in the state of California (CCR title 22) for easy disposal down the drain. See the EvaGreen Safety Report for more information.

Figure 4. EvaGreen® Dye is safer than SYBR® Green I due to the fact that it is cell membrane impermeable, and therefore cannot gain access to the DNA in the nuclei of living cells.