EvaGreen® dye is a green fluorescent nucleic acid dye with features that make the dye useful for several applications including qPCR and DNA melt curve analysis, real-time monitoring of thermophilic helicase-dependent amplification (tHDA), and capillary gel electrophoresis. The DNA-bound dye has excitation and emission spectra very close to those of fluorescein (FAM) or SYBR® Green I, making the dye readily compatible with instruments equipped with the 488 nm argon laser or any visible light excitation with wavelength in the region. EvaGreen® dye is extremely stable both thermally and hydrolytically, providing convenience during routine handling. The dye is essentially nonfluorescent by itself, but becomes highly fluorescent upon binding to dsDNA. EvaGreen® dye is nonmutagenic and noncytotoxic by being completely impermeable to cell membranes, unlike SYBR® Green I, which enters cell rapidly and is known to be a powerful mutation-enhancer (Ohta, et el. Mutat. Res. 492, 91(2001)).
The unique properties of EvaGreen® dye have made it particularly useful in quantitative real-time PCR (qPCR) applications. Compared with the widely used SYBR® Green I, EvaGreen® dye is generally less inhibitory toward PCR and less likely to cause nonspecific amplification. As a result, EvaGreen® dye can be used at a much higher dye concentration than SYBR® Green I, resulting in a high resolution signal for droplet digital PCR, real-time PCR and melt curve analysis.
EvaGreen dye and applications are covered under US patent nos. 7,803,943 and 7,776,567 and pending international patents.
Low PCR inhibition
Exhibits much less PCR inhibition than SYBR® Green I via a smart “release-on-demand” DNA-binding technology
Low PCR inhibition of the dye permits a higher dye concentration to be used for much greater qPCR and melt curve analysis signals
Nonmutagenic and noncytotoxic
Nonmutagenic and noncytotoxic by standard Ames test; completely impermeable to cell membranes
Compatible with fast PCR protocols
Minimal interference to PCR makes it possible to significantly shorten the chain extension time
Compatible with multiplex PCR
No dye migration from amplicon to amplicon when used at the recommended concentration
Unsurpassed thermal stability, chemical stability and photostability
No detectable dye decomposition in PCR buffer at 95-100°C for 48 hours; highly stable under either alkaline or acidic condition; withstands repeated freeze-thaw cycles
Spectrally similar to SYBR® Green I
Compatible with all major brand qPCR instruments and enzyme systems