Resolution Melting Dyes and probes
of High Resolution Meling (HRM) analysis is to
monitor this process happening in real-time. This is achieved by using
saturating fluorescent dye. HRM dyes are known as intercalating dyes
bind specifically and in high amount (= saturating dye, except SYBR
Green) to double-stranded DNA and when they are bound they fluoresce
brightly. In the absence of double-stranded DNA they have nothing to
bind to and they only fluoresce at a low level (estimations are between
1-4% false positive fluorescence). At the beginning of the HRM
analysis, after complete PCR amplification, there is a high level of
fluorescence in the sample because of the millions of copies of the
amplicon. But as the sample DNA is heated up and the two strands of the
DNA denaturate there is no longer any double stranded DNA present and
thus fluorescence is reduced =>
dyes or probe systems are available:
dyes are specifically designed for high-resolution melting curve
analysis to detect DNA sequence variants. The addition of LCGreen dyes
increase the melting temperature of DNA by 1-3 °C and may requre
adjustment of cycling parameters. The dyes are manufactured exclusively
by Idaho Technology and their chemical structures are unique among the
scientific and patent literature.
dyes are tailored specifically for Hi-Res Melting and have the unique
ability to detect heteroduplexes during melting analysis after PCR.
Just add the dye to your sample before PCR. LCGreen dyes are extremely
stable, do not inhibit PCR, and are "saturation" dyes that can detect
multiple PCR products in a mixture during melting analysis.
TrainingLCGreen PLUS is a new member of the dye family tailored for use
in melting instruments with 96- or 384-well microtiter plates. It has
superb fluorescence intensity and can be used with other fluorescence
based PCR detection systems such as the Roche LightCycler®. For
optimal performance, the use of a high-resolution melting instrument is
I is a dsDNA binding dye used for Hi-Res Melting curve analysis using
Idaho Technology’s HR-1™ instrument. This innovative dye is
manufactured exclusively by Idaho Technology and is designed
specifically for Hi-Res Melting analysis to detect DNA sequence
variants (SNPs, Insertions / Deletions).
LC Green Flyer
genotyping by amplicon melting analysis using LC Green.
Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ.
Clin Chem. 2003 49(6 Pt 1): 853-860
High-resolution amplicon melting analysis was recently introduced as a
closed-tube method for genotyping and mutation scanning (Gundry et al. Clin
Chem 2003;49: 396-406). The technique required a fluorescently
labeled primer and was limited to the detection of mutations residing
in the melting domain of the labeled primer. Our aim was to develop a
closed-tube system for genotyping and mutation scanning that did not
require labeled oligonucleotides.
METHODS: We studied polymorphisms in the
hydroxytryptamine receptor 2A (HTR2A) gene (T102C), beta-globin
(hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C,
I507del) gene. PCR was performed in the presence of the double-stranded
LCGreen, and high-resolution amplicon melting curves
were obtained. After fluorescence normalization, temperature
adjustment, and/or difference analysis, sequence alterations were
distinguished by curve shape and/or position. Heterozygous DNA was
identified by the low-temperature melting of heteroduplexes not
observed with other dyes commonly used in real-time PCR.
RESULTS: The six common beta-globin genotypes (AA,
AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon.
The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp
fragment that split into two melting domains. Because melting curve
acquisition required only 1-2 min, amplification and
analysis were achieved in 10-20 min with rapid cycling conditions.
CONCLUSIONS: High-resolution melting analysis of PCR products amplified
in the presence of LCGreen can identify both heterozygous and
homozygous sequence variants. The technique requires only the usual
unlabeled primers and a generic double-stranded DNA dye added before PCR
for amplicon genotyping, and is a promising method
for mutation screening.
SYTO 9 green fluorescent nucleic acid stain has been shown to stain
live and dead Gram-positive and Gram-negative bacteria, and it is a
component of the LIVE/DEAD BacLight Bacterial Viability Kits (L-7007,
SYTO ® dyes are cell-permeant nucleic acid stains that show a large
fluorescence enhancement upon binding nucleic acids. The SYTO dyes can
be used to stain RNA and DNA in both live and dead eukaryotic cells, as
well as in Gram-positive and Gramneg a tive bacteria. Available as
blue-, green-, orange- or redfluorescent dyes, these novel SYTO stains
share several important characteristics:
dyes differ from each other in one or more charac ter is tics,
including cell permeability, fluorescence enhancement upon binding
- Permeability to
virtually all cell membranes, including mammalian cells
- High molar
absorptivity, with extinction coefficients >50,000 cm-1M-1 at
visible absorption maxima
- Extremely low
intrinsic fluorescence, with quantum yields typically <0.01 when not
bound to nucleic acids
- Quantum yields
that are typically >0.4 when bound to nucleic acids
nucleic acids, excitation and emission spectra, DNA/RNA selectivity and
sensitive, and discriminating identification of Naegleria spp. by
real-time PCR and melting-curve analysis.
BS, Monis PT, Dobson PJ.
Environ Microbiol. 2006 Sep;72(9):5857-63.
Water Quality Centre, Private Mail Bag 3, Salisbury, SA 5108, Australia
amoeboflagellate genus Naegleria includes one
pathogenic and two potentially pathogenic species (Naegleria fowleri,
Naegleria italica, and Naegleria australiensis) plus numerous benign
organisms. Monitoring of bathing water, water supplies, and cooling
systems for these pathogens requires a timely and reliable method for
identification, but current DNA sequence-based methods identify only N.
fowleri or require full sequencing to identify other species in the
genus. A novel closed-tube method for distinguishing thermophilic
Naegleria species is presented, using a single primer set and the DNA
intercalating dye SYTO9 for real-time PCR and melting-curve analysis of
the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1,
ITS2). Collection of DNA melting data at
intervals produces highly informative melting curves
with one or more recognizable melting peaks, readily distinguished for
seven Naegleria species and the related Willaertia magna. Advantages
over other methods used to identify these organisms include its
comprehensiveness (encompassing all species tested to date), simplicity
(no electrophoresis required to verify the product), and sensitivity
(unambiguous identification from DNA equivalent to one cell). This
approach should be applicable to a wide range of microorganisms of
of SYTO9 and SYBR Green I for real-time polymerase chain reaction and
of the effect of dye concentration on amplification and DNA melting curve
PT, Giglio S, Saint CP.
Biochem. 2005 340(1): 24-34.
Unit, Australian Water Quality Centre, Private Mail Bag 3, Salisbury,
SA 5108, Australia.
report of the use of SYBR Green I for real-time polymerase chain
reaction (PCR) in 1997, little attention has been given to the
development of alternative intercalating dyes for this application.
This is surprising considering the reported limitations of SYBR Green
I, which include limited dye stability, dye-dependent PCR inhibition,
and selective detection of amplicons during DNA melting curve analysis
of multiplex PCRs. We have tested an alternative to SYBR Green I and
report the first detailed evaluation of the
dye SYTO9. Our findings demonstrate that SYTO9 produces highly
reproducible DNA melting curves over a broader range of dye
concentrations than does SYBR Green I, is far less inhibitory to PCR
than SYBR Green I, and does not appear to selectively detect particular
amplicons. The low inhibition and high melting curve reproducibility of
SYTO9 means that it can be readily incorporated into a conventional PCR
at a broad range of concentrations, allowing closed tube analysis by
DNA melting curve analysis. These features simplify the use of
intercalating dyes in real-time PCR and the improved reproducibility of
DNA melting curve analysis will make SYTO9 useful in a diagnostic
use of new probes and stains for improved assessment of cell viability
polymeric substances in Candida albicans biofilms.
Y, Zhang T, Samaranayake YH, Fang HH, Yip HK, Samaranayake LP.
2005 159(3): 353-60.
of Oral Biosciences, Faculty Dentistry, The Prince Philip Dental
University of Hong Kong, 34 Hospital Road, SAR, China.
genotypic cell differentiation is considered an important feature that
confers enhanced antifungal resistance in candidal biofilms. Particular
emphasis has been placed in this context on the viability of biofilm
subpopulations, and their heterogeneity with regard to the production
of extracellular polymeric substances (EPS). We therefore assessed the
utility of two different labeled lectins Erythrina cristagalli (ECA)
and Canavalia ensiformis (ConA), for EPS visualization. To evaluate the
viability of candidal biofilms, we further studied combination stains,
SYTO9 and propidium iodide (PI). The latter combination has been
successfully used to assess bacterial, but not fungal, viability
although PI alone has been previously used to stain nuclei in fungal
cells. Candida albicans biofilms were developed in a rotating disc
biofilm reactor and observed in situ using confocal scanning laser
microscopy (CSLM). Our data indicate that SYTO9 and PI are reliable
vital stains that may be used to investigate C. albicans biofilms. When
used together with ConA, the lectin ECA optimized EPS visualization and
revealed differential production of this material in mature candidal
biofilms. The foregoing probes and stains and the methodology described
should help better characterize C. albicans biofilms in terms of cell
their viability, and EPS production.
is a green fluorescent nucleic acid dye with features that make the dye
useful for several applications including qPCR, high-resolution DNA
melt curve analysis (HRM)1, real-time monitoring of thermophilic
helicase-dependent amplification (tHDA), routine solution DNA
quantification and capillary gel electrophoresis. The DNA-bound dye has
excitation and emission spectra very close to those of fluorescein
(FAM) or SYBR® dye 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,
properties of EvaGreen dye have made it particularly useful
in quantitative real-time PCR (qPCR) application. 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 more robust PCR signal.
- Very Little PCR
inhibition: Exhibit much less PCR inhibition than SYBR Green I via a
smart "release-on-demand" DNA-binding technology.
Sensitive: Low PCR inhibition of the dye
permits a higher dye concentration to be used for much greater
fluorescent signal and high-resolution melt curve analysis (HRM).
- Nonmutagenic and
noncytotoxic: Nonmutagenic and noncytotoxic by standard Ames
test; completely impermeable to cell membranes (see below).
- Compatible with Fast PCR
protocol: 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, Hydrolytical 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;
withstand repeated freeze-thaw cycles
- Spectrally similar to
SYBR Green I: Compatible with all major brand qPCR
instruments and enzyme systems
quantification using EvaGreen and a real-time PCR instrument.
Wanga, Kunsong Chena and Changjie XuCorresponding Author Contact
Information, a, E-mail The Corresponding Author
Agricultural Ministry Laboratory of Horticultural Plant Growth,
Development and Biotechnology, Huajiachi Campus, Zhejiang University,
Hangzhou 310029, People’s Republic of China
356, Issue 2, 15 September 2006, Pages 303-305
an important, frequently used technique, and inaccuracies can result in
failures with ligation, restriction, polymerase chain reaction (PCR),1
amplified fragment length polymorphism (AFLP), Southern blotting, and
other techniques. DNA is most commonly quantified using absorbance at
260 nm, but because of the existence of many impurities, this can be an
imprecise measurement and DNA levels can be more than 10 times
overestimated in some cases . Quantification by agarose gel
electrophoresis with a known amount of standard DNA  and  can
provide more accurate data, but the procedures are complicated, the
data often still are not accurate enough, and the technique is
impractical for routine or high-throughput DNA quantification .
Fluorescence spectroscopy using various DNA intercalating dyes is the
most widely accepted technique for accurate DNA quantification .
However, if the analysis is to be carried out with a fluorescence
spectrophotometer, a relatively large assay volume (e.g., 2 ml) is
required , and this is impractical for small DNA samples and
expensive dyes. Fluorescence can also be measured with a smaller volume
of DNA sample using other instruments such as fluorescent microplate
readers , microplate fluorometers  and , and
transilluminator–microplate–CCD camera systems , but the instruments
might not be readily available in most molecular biology laboratories.
of EvaGreen and the implication of its physicochemical properties
for qPCR applications.
F, Leung WY, Xin X.
Biotechnol. 2007 7(1): 76
EvaGreen (EG) is a newly developed DNA-binding dye that has recently
been used in quantitative real-time PCR (qPCR), post-PCR DNA melt curve
analysis and several other applications. However, very little is known
about the physicochemical properties of the dye and their relevance to
the applications, particularly to qPCR and post PCR DNA melt curve
analysis. In this paper, we characterized EG along with a widely used
qPCR dye, SYBR Green I (SG), for their DNA-binding properties and
stability, and compared their performance in qPCR under a variety of
conditions. RESULTS: This study systematically compared theDNA binding
of the two dyes under different conditions and had these findings: a)
EG had a lower binding affinity for both double-stranded DNA (dsDNA)
and single-stranded DNA (ssDNA) than SG; b) EG showed no apparent
preference for either GC- or AT-rich sequence while SG had a slight
preference for AT-rich sequence; c) both dyes showed substantially
lower affinity toward ssDNA than toward dsDNA and even lower affinity
toward shorter ssDNA fragments except that this trend was more
pronounced for EG. Our result also demonstrated that EG was stable both
under PCR condition and during routine storage and handling. In the
comparative qPCR study, both EG and SG exhibited PCR interference when
used at high dye concentration, as evident from delayed Ct and/or
nonspecific product formation. The problem worsened when the chain
extension time was shortened or when the amplicon size was relatively
long (>500 bp). However, qPCR using EG tolerated a significantly
higher dye concentration, thus permitting a more robust PCR signal as
well as a sharper and stronger DNA melt peak. These differences in qPCR
performance between the two dyes are believed to be attributable to
their differences in DNA binding profiles. CONCLUSION: These findings
suggest that an ideal qPCR dye should possess several DNA-binding
characteristics, including a "just right" affinity for dsDNA and low or
no affinity for ssDNA and short DNA fragments. The favorable
DNA-binding profile of EG, coupled with its good stability and
instrument-compatibility, should make EG a promising dye for qPCR and
electrophoresis of double-stranded DNA fragments using a new
fluorescence intercalating dye EvaGreen.
Sang F, Ren J.
J Sep Sci. 2006 29(9): 1275-1280.
College of Chemistry and Chemical Engineering,
Shanghai Jiaotong University, Shanghai, P. R. China.
EvaGreen is a new
DNA intercalating dye successfully used in quantitative real-time PCR.
In the present work, we firstly apply EvaGreen to the analysis of dsDNA
by CE with LIF detection. Comparisons of EvaGreen dye with the commonly
used dyes SYBR Green I and SYBR Gold were preformed in dsDNA analysis
by CE. The linear range of dsDNA using EvaGreen was slightly wider than
that using SYBR Gold and SYBR Green I, and the detection limits of
dsDNA were not significantly different for the three dyes. Good
separations of dsDNA fragments were obtained using the three dyes.
Reproducibility of migration time and the peak area of dsDNA fragments
with EvaGreen were better than those for SYBR Green I and SYBR Gold.
The RSD values were 0.24-0.27% for migration time and 3.45-7.59% for
peak area within the same day, 1.35-1.63% for migration time and
6.72-12.05% for peak area for three days. Our data demonstrated that
EvaGreen is well suited for the dsDNA analysis by CE with LIF detection.
with SYBR Green (by Corbett Life
Moreover, the use
SYBR® Green 1 (SYBR) for HRM is actively discouraged by some
authors (Wittwer et al 2003; Reed, Kent & Wittwer, 2007). Liew
et al (2004) state that SYBR can only be used for HRM after
substantial modification of the protocol (including the use of
GC-clamps, triple primers, and allele-specific PCR). By contrast, we
and others (Price et al 2007; Pornprasert et al 2008) have
found SYBR to
be a very successful dye for HRM analysis that does not require any
protocol modifications. We unhesitatingly recommend its use with a
SYBR stems from early evidence collected on instrumentation that was
not ideal for the task. Original assertions were based on experiments
done with a pre-HRM era LightCycler™ capillary-based real-time analyzer
(Roche Molecular Systems) using templates that included a low mass DNA
size ladder (Wittwer et al 2003;
Liew et al 2004). The
reason SYBR was less successful than LCGreen was “not entirely clear”
at the time, but a “dye redistribution” hypothesis was suggested (Wittwer et al 2003). According
to this hypothesis, SYBR dye releases from low-temperature duplexes
during melting and dynamically re-intercalates into neighboring
duplexes that melt at higher temperature. This mechanism became the
basis for the “dye saturation model” (Wittwer et al 2003, Liew et al 2004). According to this
model, if sufficiently high concentrations of dye are used such that
all binding positions on the DNA are occupied (i.e. saturated), then
dye redistribution effects are minimized and greater melting curve
resolution can be achieved. So-called “saturating dyes” were defined as
those that can be used at concentrations sufficiently high to saturate
all DNA binding sites without inhibiting the PCR. This model formed the
basis for IP protection and a patent by
Witter et al and Idaho Technology.
In spite of the
saturation model, mounting evidence clearly shows that it is not valid
on the Rotor-Gene 6000 HRM instrument. This is true for a range of
dyes, including SYBR (manuscript in preparation). In fact, SYBR used at
standard non-saturating concentrations is highly suitable for HRM
analysis, as illustrated below for the detection of a Class 4 SNP. We
therefore urge users not to dismiss the use of SYBR for HRM.
dye levels are not required for HRM on the Rotor-Gene 6000 (when
apparently required on other instrument systems) is not clear. We have
limited experience with competing instruments, however it must be noted
that the centrifugal rotary format employed by the Rotor-Gene is
distinctly different to other HRM instrumentation. Importantly, the
Rotor-Gene has 25–50 times the thermal precision of other instruments
and the shortest, most sensitive, and most uniform optical path. It
also averages multiple readings for each data point reported at each
discrete programmed thermal setpoint in a HRM. Surprisingly, Reed et al
(2007) claim that the Rotor-Gene can only “approach high-resolution
data quality by melting at slower rates”. Ironically, it may be that
the slightly slower and more deliberate thermal stepping used by the
Rotor-Gene is partly why it achieves superior HRM results without the
need for a “saturating dye”.
Genotyping a Class 4 SNP
by high resolution melt (HRM) using SYBR Green I
Alister Kwok, Brant Bassam, and Valin Reja, August 2007
SensiMix HRM has been
designed for High Resolution Melt (HRM) analysis on the Rotor-Gene
6000. To learn more about the applications of HRM and to see some
data from our mix, please click
HRM optimised mix with a separate vial of EvaGreen dye.
Ultra-high sensitivity: detects class 4 (A/T) SNP mutations.
Comes in 250, 500 and 2000 (25µl) reaction packs.
HRM™ demo kit containing this mix is available to Corbett Life Science
for qPCR & HRM
Odinsgatan 28, 41103
Chromofy is a
monomeric asymmetric cyanine dye, developed by TATAA
Biocenter for use in qPCR applications. The dye has absorbance and
emission wavelengths that are suitable for the FAM/SYBR channel on most
common real-time PCR instruments. When binding to dsDNA Chromofy shows
a very strong fluorescence increase.
in High-resolution melting
melting is a post-PCR analysis that discriminates
different samples on the basis of their melting temperature. Chromofy
and HRM can be used for genotyping, either with ro without unlabelled
probes and for methylation analysis. Methylation analysis is performed
after bisulfite treatment of the DNA samples which converts
unmethylated cytosines to uracils while methylated cytosines remain
intact. During subsequent PCR the uracils will be substituted for
thymines giving rise to a Tm difference between methylated and
un-methylated samples, which is seen as varying heights of the plateu
in the normalized HRM data. Using Chromofy your assay can be sensitive
down to 1 % methylated DNA in un-methylated background.
download Chromofy user
sequence-specific probes and DNA binding dyes in real-time PCR for
specific nucleic acid quantification and melting curve analysis
Lind1,2, Anders Ståhlberg2,3, Neven Zoric2, and Mikael Kubista1,2
Chalmers University of Technology, Gothenburg, 2 TATAA Biocenter,
Gothenburg, and 3 Lund University, Lund, Sweden
40:315-319 (March 2006)
real-time PCR, one often has to choose between using a
sequence-specific probe and a nonspecific double-stranded DNA (dsDNA)
binding dye for the detection of amplified DNA products. The
sequence-specific probe has the advantage that it only detects the
tar-geted product, while the nonspecific dye has the advantage that
melting curve analysis can be performed after completed amplification,
which reveals what kind of products have been formed. Here we present a
new strategy based on combining a sequence-specific probe and a
nonspecific dye, BOXTO, in the same reaction, to take the advantage of
both chemistries. We show that BOXTO can be used together with both
TaqMan® probes and locked nucleic acid (LNA) probes without
interfering with the PCR. The probe signal reflect formation of target
product, while melting curve analysis of the BOXTO signal reveals
primer-dimer formation and the presence of any other anomalous products.
an unsymmetric cyanine dye developed by TATAA Biocenter for use in qPCR
dye has absorbance and emission wavelengths that can be detected on
the FAM channel on most common real-time PCR platforms, and shows a
strong fluorescence increase when bound to dsDNA. BEBO can be used as
an unspecific dye for real-time PCR applications or other applications
where staining of dsDNA is wanted.
new minor groove binding asymmetric cyanine reporter dye for real-time
Bengtsson, H. Jonas Karlsson, Gunnar Westman and Mikael Kubista*
of Chemistry and Bioscience, Chalmers University of
Technology 41296 Goteborg and
Odinsgatan 28, 41103 Goteborg, Sweden
Acids Research, 2003, Vol. 31, No. 8 e45
The minor groove
asymmetric cyanine dye 4-[(3-methyl-6- (benzothiazol-2-yl)-
-2-methylidene)]- 1-methyl-pyridinium iodide (BEBO) is tested as
sequence nonspeciÆc label in real-time PCR. The
Fluorescence intensity of BEBO increases upon binding to double-stranded
emission to be measured at the end of the elongation phase in the PCR
cycle. BEBO concentrations
between 0.1 and 0.4 mM generated sufÆcient Øuorescence
inhibiting the PCR. A comparison with the commonly used reporter dye
SYBR Green I shows that the two dyes behave similarly
in all important aspects.
How does the service
"On demand" HRM assays for any variations in any DNA
sequence. Simply give us the details of your variation of
interest and our expert team will professionally design the best
possible primers that cater for your individual requirements. We
then scientifically validate the assay on biologically derived gDNA in
our own laboratory and package them for you at optimal concentrations
for HRM analysis. The assays arrive with you in less than three
weeks and no further optimisation by you is required.
What is an HRM raZor probe?
Custom designed HRM assays that come with a Razor
probe are the highest specification HRM assay available. An
especially modified DNA probe targets the site of the SNP or DNA
variation of interest. HRM analysis of the probe/target melting
adds huge power the quality of data achieved. We guarantee the
SNP typing power of these assays.