The very best dye for qPCR and HRM

Figure 1. Amplifications of a Myc gene fragment (75 ng huDNA input) (ABI) using EvaGreenTM (red), SYBR® GreenERTM (green) (Invitrogen) and Power SYBR® (yellow) (ABI), respectively. All reactions were run in duplicate on an ABI 7900 instrument using the Universal cycling protocol (15 s at 95 oC and 60 s at 60 oC).

EvaGreen® is a DNA-binding dye with many features that make it a superior alternative to SYBR® Green I for quantitative real-time PCR (qPCR). Apart from having similar spectra, EvaGreenTM has three important features that set it apart from SYBR® Green I.

First, EvaGreenTM has much less PCR inhibition than SYBR® Green I. As a result, qPCR employing EvaGreenTM may be run using a fast PCR protocol (Figure 2). Moreover, EvaGreenTM can be used at a relatively high concentration, which results in a significantly higher PCR signal than that with SYBR® Green I (Figure 1). The relatively high EvaGreenTM concentration also eliminates socalled "dye redistribution" problem, making EvaGreenTM suitable for both PCR multiplexing and high-resolution melt curve analysis (HRM) (Figure 3), a technique that is gaining popularity in genotyping and heteroduplex detection following PCR.1 Dye redistribution, which is caused by low dye concentration, is an unsolvable problem for SYBR® Green I because its high tendency to inhibit PCR requires that the dye be used at a very low concentration. Thus, SYBR® Green I is not suitable for either multiplex PCR or HRM.1,2 Moreover, dye redistribution may make SYBR® Green I unreliable even for regular melt curve analysis because low-melting DNA species may simply not have been detected.2,3

Second, EvaGreenTM is an extremely stable dye. Under the normal conditions of storage, handling and PCR operation, the dye is virtually indestructible. The dye in a buffer can be safely stored at room temperature or in a refrigerator, or can be subject to multiple freeze-thaw cycles. On the other hand, SYBR® Green I is known to be unstable and the decomposed dye is even more inhibitory to PCR.4

Third, we have made EvaGreenTM a much safer dye than SYBR® Green I by rendering it cell membrane- impermeable (Figure 5). Indeed, an independent laboratory test has shown that EvaGreenTM is both nonmutagenic and noncytotoxic.5 In comparison, although SYBR® Green I is only weakly mutagenic by itself, it has been shown to be a potent mutation-enhancer by possibly inhibiting the natural DNA repairing mechanism in cells.6 In view of the widespread practice of qPCR, we believe that safety of the dye should be an important consideration.

We offer several EvaGreen® products. EvaGreen® Master Mix (#31003) contains everything you need except for the primer set to amplify your target DNA. EvaGreen® Master Mix is also suitable for high resolution melt curvem(HRM) analysis. EvaGreen® 20X (#31000) is a solution containing optimally adjusted dye concentration that can be diluted 20 times to make your own EvaGreen® master mix.

  • Herrmann, et al. Clin. Chem. 52(3), 494(2006).
  • Wittwer, et al. Clin. Chem. 49(6), 853(2003).
  • Giglio, et al. Nucleic acids Res. 31(22), e136(2003).
  • Karsai, et al. BioTechniques 32(4), 790(2002).
  • A full safety report is available at Biotium website
  • Ohta et al. Mut. Res. 492, 91(2001).


Highly sensitive Produces the most robust PCR signal when used at the recommended concentration.

Low PCR inhibition Exhibits much less PCR inhibition than SYBR Green I via a smart "release-ondemand" DNA-binding technology.

Compatible with Fast PCR protocol Minimal interference to PCR makes it possible to significantly shorten the chain extension time.

Excellent for HRM application Lack of "dye redistribution" problem makes it compatible with post-PCR high-resolution melt curve analysis (HRM) in a closed-tube format.

Compatible with multiplex PCR No dye migration from amplicon to amplicon when used at the recommended concentration.

Extremely stable Simply indestructible under most biochemical conditions. Can be stored at room temperature and be subject to repeated freeze-thaw cycles.

Spectrally similar to SYBR® Green I Compatible with all major brand qPCR instruments.

Nonmutagenic and noncytotoxic

EvaGreenTM Is Compatible with Fast PCR Protocol
Figure 2. Comparison of EvaGreenTM Master Mix and Power SYBR Master Mix (ABI) in GAPDH amplification. The two-stage Universal Cycling condition was used with 15 seconds at 95 oC and a varying amount of annealing/extension time (60, 40, 20 and 10 seconds, respectively) at 60 oC. Each horizontal bar represents the Ct distribution of 8 repeat experiments. The data shows that reduction in annealing/extension time from 60 s to 10 s resulted in only about one cycle delay in Ct with EvaGreen Master Mix, but as many as 5-6 cycle delay in Ct and poor reproducibility with Power SYBR Master Mix.

EvaGreenTM Can Be Used for High-resolution Melt Curve Analysis
Figure 3. High-resolution DNA melt curve (HRM) analysis using EvaGreenTM on Rotor Gene 6000 clearly distinguishes three different genotypes: mutant (red), heterozygous (purple) and wild (blue). (Data contributed by Corbett Research).

Table 1. EvaGreen® Product List
Cat.# Product Name Unit Size
31003 Fast EvaGreen®Master Mix for qPCR and HRM 2x1 mL
31003-1 Fast EvaGreen®Master Mix for qPCR and HRM 5x1 mL
31003-2 Fast EvaGreen®Master Mix for qPCR and HRM 50x1 mL
31000 EvaGreen® dye, 20x in water 5x1 mL
31001 EvaGreen® qPCR 2X Basic Mix 3x1.7 mL
31004 EvaGreen® qPCR 2X Basic Mix for Hot Start Taq 3x1.7 mL
EvaGreenTM Can Be Optimally Excited by the 488 Ar Laser or Blue LED
Figure 4. Excitation and emission spectra of EvaGreenTM in the presence of dsDNA in PBS buffer.

EvaGreenTM Is Safer than SYBR® Green I by Being Impermeable to Cell Membranes
Figure 5. HeLa Cells incubated with either SYBR® Green I (1.2 μM, left panel) or EvaGreenTM (1.2 μM, right panel) at 37 oC were followed by fluorescence microscopy. No cell staining was observed with EvaGreenTM during 30 minutes of incubation, suggesting that EvaGreenTM did not cross cell membranes. However, significant cellular staining was observed with SYBR® Green I in less than 5 minutes of incubation (data not shown). After 30 minutes, SYBR® Green I stained cell nuclei intensely green (left panel). The rapid cellular uptake of SYBR® Green I, coupled with the dye's known potent mutation-enhancing ability (Ohta et al. Mutation Research 492, 91(2001)), makes the dye a potential hazard during routine PCR operation. EvaGreenTM, on the other hand, appears to be completely membrane-impermeable, which may be at least partially responsible for its nonmutagenicity and noncytotoxicity as confirmed by standard Ames test (A full safety report on EvaGreenTM can be downloaded at Biotium website).

EvaGreen® is for end-users only. To license the technology for commercial use, please contact Biotium. To practice qPCR and/or HRM, you may need additional licenses from ABI, Roche and any other relevant patent holders.

Another breakthrough qPCR technology, AllGloTM Probes, may also be licensed through Biotium. Developed by Allelogic Biosciences Corp, AllGloTM Probes are superior alternatives to TaqMan®-MGB probes by having better discrimination power in detecting single mutation sites, better signal strength and much simpler design.