|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).|
|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||EvaGreen® active form||EvaGreen-ADN complex|
EvaGreen® Dye binds to dsDNA via a novel “release-on-demand” mechanism.
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.
|Fast Plus EvaGreen® qPCR Master Mix – No ROX #31020
|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 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|
|Order the 2-Color version ROX #31042|
|Order the 2-Color version ROX #31041|
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)|
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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.