The quantitative real-time polymerase chain reaction (qPCR) technique is widely used for scientific, clinical, diagnostic, or quality control purposes. The applications of qPCR include gene expression analysis, mutation detection, genotyping, DNA detection, and quantification (like pathogens). Logo Biotium
Although Taqman fluorescent probes confers high specificity and sensitivity to assays, nonspecific detection based on DNA-binding dyes (i.e. EvaGreen®, SYBR® Green I) offers versatility in qPCR assays.

Despite their wide use and good performance, used DNA-binding dyes have in some instances a safety concern (DNA binder are mutagenic). Lets explain why EvaGreen® overcomes these discrepencies compared with SYBR® Green I . And how Evagreen keeps advantage over Taqman probes in many applications. Finally certain dyes allow qPCR to perform in viable cells !

1. What is the difference between EvaGreen® and SYBR® Green?

EvaGreen® dye is spectrally similar to FAM or SYBR® Green I, which means no change in optical settings is required for using an EvaGreen®-based master mix. However, there are several differences between the dyes.

a. Unique principle:

EvaGreen® dye operates by a novel “release-on-demand” mechanism:

EvaGreen inactive form Flèches EvaGreen active form Flèches EvaGreen-DNA complex
EvaGreen® inactive form EvaGreen® active form EvaGreen-ADN complex

EvaGreen® Dye binds to dsDNA via a novel “release-on-demand” mechanism.


b. Safer:

EvaGreen® dye is the first and only PCR dye to date designed to be safe and environmentally safe. Pictos sans danger pour l'environnement
Independent labs have confirmed that EvaGreen® dye is non-mutagenic, non-cytotoxic, and safe to aquatic life for direct disposal in the drain. Other commercial PCR dyes enter into cells in a matter of minutes, while Evagreen® does not. SYBR® Green I, for example, has been even shown to be environmentally more toxic than ethidium bromide, a well-known mutagen [Ohta T.]. For details, download the EvaGreen® Dye Safety Report.



c. Lower background fluorescence:

EvaGreen® dye has less background than SYBR® Green I due to its novel “release-on-demand” DNA-binding mechanism..

d. Brighter and non-inhibitory:

EvaGreen® is less inhibitory in the PCR reaction than SYBR® Green, permitting the use of a saturating dye concentration for maximal signal and better high-resolution DNA melt analysis..


e. Dye stability

EvaGreen® dye is very stable both during storage and under PCR conditions [Nowaldy]. 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.

f. Digitale PCR

EvaGreen® dye is currently the only qPCR DNA-binding dye to be used in droplet digital PCR (ddPCR).[Dermott]


2. Peace of Mind plus PCR Performance

EvaGreen® qPCR Master Mix is formulated for fast cycling PCR parameters, but can also be used with regular cycling protocols. It will make it easy for you to make sure your reactions are set up correctly the first time, every time. Furthermore several versions are available to fit your need : with a ROX internal reference, and a tracking dye to avoid reagent distribution mistakes.

  • Fast Plus EvaGreen® qPCR Master Mix
    contains Evagreen® qPCR dye and Cheetah™ Taq hotstart DNA polymerase. It is suitable for qPCR using a fast cycling protocol.
    Fast Plus Evagreen® qPCR Master Mix is a 2X master mix, and is supplied with a separate vial of ROX reference dye, for use with no ROX, low ROX, or high ROX instruments.
Designation Technical sheet
Fast Plus EvaGreen® qPCR Master Mix – No ROX #31020
Picto Datasheet
Fast Plus EvaGreen® qPCR Master Mix – Low ROX #31014 – – –
Fast Plus EvaGreen® qPCR Master Mix – High ROX #31015 – – –


  • 2X Forget-Me-Not™ EvaGreen®
  • qPCR Master Mix
    Improved version with a low concentration of an additional blue tracking dye for visual control of correct distribution.
    Prevent wasted samples and other costly mistakes with our Forget-Me-Not™ light blue master mix.

Forget-Me-Not™ EvaGreen® qPCR Master Mix

Forget-Me-Not™ EvaGreen® qPCR Master Mix contains a low concentration of an inert blue dye, which allows the user to visually distinguish wells containing reaction mix from empty wells, and can thereby reduce pipetting errors, saving time and reagents.

Forget-Me-Not™ EvaGreen® qPCR Master Mix – low ROX #31045
Forget-Me-Not™ EvaGreen® qPCR Master Mix – high ROX #31046


  • 2X Forget-Me-Not™ EvaGreen® qPCR Master Mix (2-color tracking system)
    Distinguish master mix or template wells ! Prevent wasted samples.
Forget-Me-Not™ EvaGreen® qPCR Master Mix (2-color tracking system)
Designation Technical sheet
Order the 2-Color version ROX #31042 Picto Datasheet
Order the 2-Color version ROX #31041 Picto Datasheet


qPCR also perform in living cells!

Using viability dye (i.e. propidium monoazide, PMA or PMAxx™), qPCR can detect selectively viable cells[Knut Rudi]. The dye binds on the DNA of dead cell, and is covalently attached after photoactivation. The main application is sensitive detection of bacterial pathogen[Zhao].

Designation FluoProbes PMA #BZ9340 Technical sheet
Order Propidium monoazides (PMAs) Picto Datasheet


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References :

Ohta T. et al., Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 492:1–2, p.91-97 (2001)

McDermott, et al. Multiplexed target detection using DNA-binding dyechemistry in droplet digital PCR. Anal Chem 85, 11619-11627 (2013). 10.1021/ac403061n (EvaGreen multiplex in Biorad ddPCR)

Nowadly C. et al; Characterization of the Effects of Heat Stress on the DNA-Intercalating Dye EvaGreen for Potential Use With the Joint Biological Agent Identification and Diagnostic System Craig D. ; Mil Med. 179(6):626-32) (2014)

Knut Rudi et al., Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples – Appl. Environ. Microbiol. vol. 71 no. 2 1018-1024 (2005)

Zhao L. et al., Improved quantitative detection of VBNC Vibrio parahaemolyticus using immunomagnetic separation and PMAxx-qPCR, Food Control, 110:106962 (2020)


EvaGreen Dye, Cheetah Taq and PMAxx are covered under US and international patents.
Forget-Me-Not, PMAxx, EvaGreen Dye and Cheetah Taq are trademarks of Biotium.
SYBR is a registered trademark of Thermo Fisher Scientific.
TaqMan is a registered trademark of Roche Molecular Systems.