EvaGreen

A PCR Dye Safe to the Environment

February 07, 2011

FEATURES E vaGreen® dye is a next-generation DNA- conformation that is capable of binding to DNA to
emit fluorescence. The chemical equilibrium pro-
binding dye with features ideal for use in quan-
 Environmentally safe titative real-time PCR (qPCR) and many other vides a unique mechanism to continuously supply
applications. Biotium scientists designed the dye the active form of the dye from the "reserve" (i.e.,
Non-mutagenic, non-cytotoxic and safe to
aquatic life for safe handling and easy disposal by taking into consideration of several essential the dye in looped conformation), as more DNA is
down the drain. dye properties relevant to PCR, including PCR formed during a PCR process. Consequently, an
inhibition, safety, stability and fluorescence spectra EvaGreen® master mix can be formulated with
 Superior for qPCR and isother- of the dye. The results of our effort is a dye superior relative high dye concentration to maximize fluo-
mal amplification to SYBR® Green I and other commercial PCR or rescence signal without PCR inhibition, making the
Far brighter than SYBR® Green I for detecting high-resolution melt curve (HRM) dyes.1-4 mix suitable for both qPCR and HRM applications
amplification due to novel "release-on-demand" (Figures 2).* Moreover, the EvaGreen® dye in the
PCR Performance: A PCR dye emits fluorescence
DNA-binding mechanism. mix is sufficiently concentrated to serve as a DNA
by forming a dye-DNA complex. The interaction
gel stain such that PCR product can be directly
 Unrivaled DNA melt curve with DNA inevitably leads to some PCR interfer-
analyzed by gel electrophoresis without the need
performance ence by a number of ways, including making ds-
for another gel stain. Visit Biotium website for more
DNA more difficult to melt, promoting primer-dimer
Low PCR inhibition permitting use of saturation information on Biotium's optimally formulated Fast-
dye concentration for maximal signal and high- formation and/or mis-priming, and dye acting as
Plus EvaGreen® master mix products.
resolution DNA melt analysis.* "road bumps" to slow down the chain extension
reaction. PCR inhibition by the dye can be par- Dye Safety: Another major advantage of
 Serving both as a qPCR dye ticularly serious EvaGreen® dye over
and a DNA gel stain at the early stage other PCR and HRM
DNA
Electrophoretically separated PCR product visu- of PCR, where dyes is its safety.
alized directly via a UV box without the need for the dye-to-DNA EvaGreen ® dye is
another gel stain. EvaGreen, EvaGreen,
ratio is high. On inactive form active f orm
EvaGreen-DNA complex
the first and only
the other hand, PCR dye to date
 Compatible with multiplex PCR
having sufficient Figure 1. EvaGreen® dye binds to dsDNA via a "release-on-demand" designed to be en-
Lack of dye migration from amplicon to mechanism.
amount of dye in vironmentally safe.
amplicon enabling detection of multiple PCR
products by melt curves. a master mix is Very few PCR dyes
important for generating good signal. Thus, an have been thoroughly studied for their safety de-
 Extremely stable optimal dye concentration must be used in order to spite the increasing use of PCR in research and
Perfectly stable during storage or under PCR attain reliable PCR performance. For many current diagnostics and the fact that DNA-binding dyes
condition. PCR dyes, such as SYBR® Green I, the optimal are inherently dangerous due to their potential to
dye concentration can be quite low, which limits cause mutation. Thus, handling and disposal of
 Applicable in other applica- PCR signal and also makes the dyes unsuitable PCR master mixes can be a health and environ-
tions
for high-resolution melt curve (HRM) analysis.4 mental issue. Indeed, SYBR® Green I is found to
Can be used as a general dsDNA-binding dye Furthermore, a master mix with low SYBR® Green be even more environmentally toxic than ethidium
for DNA quantitation in solution, capillary gel
concentration may fail to detect multiple amplicons bromide, one of the best known mutagen.6 SYBR®
electrophoresis and more.
by melt peaks due to dye migration from small am- Green I has been suggested to interfere with the
plicons to large amplicons, giving the false result natural DNA-repair mechanism in cells and as a
* Practicing HRM mar require a license from Idaho
of a clean single amplicon for a PCR that may in result it potentiates genotoxicity of chemicals as
Technologies, Inc.
effect produces several products.5 well as DNA damage by UV light. Although no
safety data are available on other PCR and HRM
EvaGreen® dye is designed using a novel
dyes (e.g., SYTO9, LC Green, BRYT Green and
concept of DNA binding via "release-on-demand"
ResoLight), those dyes are all known to enter cells
mechanism (Figure 1). The dye is constructed of
in a matter of minutes, thus posing potential geno-
two monomeric DNA-binding dyes linked by a flex-
toxicity risk. With this in mind, Biotium’s scientists
ible spacer. In the absence of DNA, the dimeric dye
designed EvaGreen® dye to be cell membrane
assumes a looped conformation that is inactive in
impermeable by increasing the molecular size and
DNA binding. When DNA is available, the looped
charge of the dye (Figure 3). Because EvaGreen®
conformation shifts via an equilibrium to a random
dye is denied the chance to interact with genomic DNA in living cells, Much Safer due to Impermeability to Cell membranes
it is made much safer than the other dyes. Independent laboratory
SYBR Incubation
tests have confirmed that EvaGreen® is nonmutagenic, noncytotoxic Green I EvaGreen time (min)
and safe to aquatic life. The dye has passed environmental hazard-
ous waste regulation in the state of California (CCR title 22) for easy
disposal down the drain. (Visit Biotium website for full EvaGreen® dye
5 min
safety report).

Dye Stability: EvaGreen® dye is perfectly stable both during storage

and under PCR condition. SYBR® Green I, on the other hand, is known
to degrade following multiple freeze-thaw cycles and under PCR condi-
tion. Moreover, decomposed SYBR® Green I is reported to be even 30 min
more inhibitory to PCR.7 Thus, when assessing the performance of
an EvaGreen-based master mix, you can eliminate the stability of the
dye as a variable.

Spectral Compatibility: EvaGreen® dye is spectrally similar to FAM

30 min,
or SYBR® Green I, which means no change in optical setting for using long photo-
an EvaGreen-based master mix (Figure 4). exposure

Other applications: EvaGreen® dye has been applied in numerous

other applications, such as isothermal amplification, capillary gel
Figure 3. Comparison of cell membrane permeability between EvaGreen® dye and SYBR®
electrophoresis, DNA quantitation in solution and selective detection Green I. HeLa cells were incubated with SYBR® Green I (1.2 µM) or EvaGreen® dye (1.2
of dead cells in cell viability tests. µM) at 37 oC. Photographs were taken following incubation for 5 and 30 minutes. SYBR®
Green I entered cells rapidly while EvaGreen® dye appeared membrane-impermeable as
Biotium offers several EvaGreen® dye-based products, including evident from the absence of cell nuclear staining. Image taken with long photo-exposure
time revealed that EvaGreen® dye only associated with cell membranes.
stand-alone EvaGreen dye and EvaGreen® dye master mix products
(See Table 1). Biotium's EvaGreen® dye technologies are available for
licensing.

Spectrally Compatible with Existing Instruments

References:
1. Khan, et al. Detection of aacA-aphD, qacEδ1, marA, floR, and tetA genes from multidrug-resistant
bacteria: comparative analysis of real-time multiplex PCR assays using EvaGreen® and SYBR®
Green I dyes, Molecular and Cellular Probes (2011), doi: 10.1016/j.mcp.2011.01.004.
Excitation

2. Cheng, et al. Detection of hemi/homozygotes through heteroduplex formation in high-

Emission
resolution melting analysis, Anal. Biochem. 410, 158(2011).
3. White, et al. Methylation-sensitive high-resolution melt-curve analysis of the SNRPN gene as
a diagnostic screen for Prader-Willi and Angelman Syndromes. Clin. Chem. 53, 1960 (2007).
4. Mao, et al. Characterization of EvaGreen Dye and the implication of its physicochemical
properties for qPCR applications. BMC Biotechnology 7, 76 (2007).
5. Giglio S, et al. Demonstration of preferential binding of SYBR Green I to specific DNA frag-
ments in real-time multiplex PCR. Nucleic Acids Res. 31(22), e136(2003). Wavelength (nm)
6. Ohta, et al. Ethidium bromide and SYBR Green I enhances the genotoxicity of UV-irradiation
and chemical mutagens in E. coli, Mut. Res. 492, 91(2001). Figure 4. Excitation and emission spectra of EvaGreenTM in the presence of dsDNA
7. Karsai, et al. Evaluation of a homemade SYBR green I reaction mixture for real-time PCR in PBS buffer.
quantification of gene expression. BioTechniques 32(4), 790(2002).

Table 1. EvaGreen® dye products

Product Group Cat # Packaging Size

Unrivaled PCR Performance
4
EvaGreen dye, 20X in H2O 31000 5 X 1 mL
Company A 200 ng
Company A 20 ng
Fast-Plus EvaGreen Master Mix (no ROX) 31020 200 rxn (2 X 1 mL)
Company A 2 ng
3 Company A 200 pg
Company A 20 pg
Fast-Plus EvaGreen Master Mix with Low ROX 31014 200 rxn (2 X 1 mL)
Company A NTC
Company Q 200 ng Fast-Plus EvaGreen Master Mix with High ROX 31015 200 rxn (2 X 1 mL)
Company Q 20 ng
Company Q 2 ng
AFU

2
Company Q 200 pg
Company Q 20 pg
Company Q NTC
Fast EvaGreen 200 ng

1
Fast EvaGreen 20 ng
Fast EvaGreen 2 ng
Biotium, Inc.
Fast EvaGreen 200 pg 3159 Corporate Place
Fast EvaGreen 20 pg
Fast EvaGreen NTC Hayward, CA 94545
0 www.biotium.com
15 20 25 30 35 40
Cycle Number

Figure 2. Comparison among Fast-Plus EvaGreen® master mix from Biotium and two fast SYBR®
Green master mixes from two leading companies (company A and company Q) under similar * Practicing HRM may require a license from Idaho Technologies, Inc.; SYBR, ResoLight, LC Green
condition. The inset is an enlarged view of the area near the baseline for better viewing the and BRYT Green are trademarks of Invitrogen, Roche, Idaho Technologies and Promega, respectively;
curve patterns of the much weaker signals of the two SYBR-based master mixes. Amplicon: EvaGreen technologies are covered by US patent Nos 7,601,498, 7,776,567 and other pending US and
international patents.
ATPG fragment of human genomic DNA; instrument: ABI 7900 Fast.