Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis.

Abstract

Following the initial 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 intercalating 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 context.

Full text

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ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 340 (2005) 24–34
www.elsevier.com/locate/yabio
0003-2697/$ - see front matter. Crown copyright  2005 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2005.01.046
Comparison of SYTO9 and SYBR Green I for real-time polymerase
chain reaction and investigation of the eVect of dye concentration
on ampliWcation and DNA melting curve analysis
Paul T. Monis¤, Steven Giglio, Christopher P. Saint
Microbiology Unit, Australian Water Quality Centre, Private Mail Bag 3, Salisbury, SA 5108, Australia
Received 28 October 2004
Abstract
Following the initial 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 limita-
tions of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons dur-
ing DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the Wrst detailed
evaluation of the intercalating dye SYTO9. Our Wndings 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 reproduc-
ibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.
Crown copyright  2005 Published by Elsevier Inc. All rights reserved.
Keywords: PCR; DNA melting curve analysis; Intercalating dye
Real-time PCR is rapidly becoming a standard
method in many diagnostic and research laboratories.
The market for this technology continues to expand and
there have been many probe-based systems developed to
meet this demand, including Taqman probes [1], molecu-
lar beacons [2], FRET1 probes [3], Scorpions [4], and
iFRET [5]. An alternative to probe-based detection is the
use of Xuorescent double-stranded DNA (dsDNA)-spe-
ciWc intercalating dyes. Such a dye, ethidium bromide,
was used in the Wrst description of real-time PCR by
Higuchi et al. [6,7]. Since then, a relatively small number
of dyes, including YO-PRO-1 [8], SYBR Green I [9,10],
BEBO [11], and LC Green [12], have been evaluated for
use in real-time PCR applications. While probe-based
detection methods continue to be developed and
improved, progress for intercalating dyes has been rela-
tively slow.
SYBR Green I has become the preferred choice for
real-time PCR because it is cost eVective compared to
probe-based systems, allows the generic detection of
ampliWed DNA, and can be used to diVerentiate DNA
by DNA melting curve analysis [9,10]. However, SYBR
Green I also has disadvantages that limit its ease of use.
It cannot generally be used in standard PCR buVers
without further optimization of conditions and the
inclusion of additional reagents to improve reaction
eYciency, such as DMSO [13], bovine serum albumin,
¤Corresponding author. Fax: +61 8 8259 0228.
E-mail address: paul.monis@sawater.com.au (P.T. Monis).
1Abbreviations used: FRET, Xuorescence resonance energy transfer;
DMSO, dimethyl sulfoxide.

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Real-time PCR and melting curve analysis using SYTO9 / P.T. Monis et al. / Anal. Biochem. 340 (2005) 24–34
25
and Triton X-100 [14]. Depending on the reaction condi-
tions, the dye also appears to be inhibitory to PCR in a
concentration-dependent manner [9,15] which can be
overcome by increasing the concentration of MgCl2 in
the reaction [14,15]. The degradation products of the dye
have also been reported to be inhibitory to PCR [14].
DNA melting curve analysis using SYBR Green I is
widely applied for the identiWcation of a variety of
organisms (e.g., Leptonema [16], Borellia [17,18], Legion-
ella [19], and Cryptosporidium [20]). However, the melt-
ing temperature (Tm) obtained using this dye can be
aVected by the concentration of dye [10] and the concen-
tration of DNA [21]. For example, in the study of Ririe
et al. [10] a 3 log-fold diVerence in the number of starting
copies of target DNA used in real-time PCR resulted in
a Tm diVerence of more than 1°C for the same amplicon.
A further limitation of SYBR Green I is that it
appears to have limited application for the analysis of
multiplex PCR. In a study by Giglio et al. [22], multiplex
PCRs for Vibrio cholerae and Legionella pneumophila
were analyzed by DNA melting curve analysis using
SYBR Green I. It was found that only one amplicon
could be detected by melting curve analysis but that
both amplicons were being ampliWed as determined by
agarose gel electrophoresis. This eVect appeared to be
related to the relative concentrations of the ampliWed
DNA and dye because time-based analysis demon-
strated that both amplicons could be detected by melting
curve analysis after 20 cycles of PCR but only one
amplicon could be detected after 40 cycles of PCR.
The SYTO family of dyes have many properties that
make them useful for labeling live cells, including good
membrane permeability, low cytotoxicity, and enhanced
Xuorescence in the presence of DNA (US patent
5,436,134 [23]). Although they are ostensibly DNA-bind-
ing dyes, their low toxicity suggests that they may not
interfere with cellular processes, such as DNA and RNA
synthesis, at the concentrations that are used for staining
cells. Little information with regard to their speciWcity
for dsDNA versus single-stranded DNA (ssDNA) or
their suitability as dyes for real-time PCR is available.
Evaluation of the mode of action of the BacLight Live/
Dead kit (Molecular Probes) in our laboratory suggested
that SYTO9 is a dsDNA-speciWc dye. These factors,
together with the similarity in excitation/emission max-
ima in SYTO9 and the FAM channel of the Rotor Gene
3000, have led us to select SYTO9 for evaluation in real-
time PCR.
We report herein the novel use of SYTO9 in real-time
PCR. This dye supports PCR ampliWcation over a wide
range of dye concentrations and produces robust DNA
melting curves that are not aVected by DNA concentra-
tion. In addition, we investigated the inhibitory eVect of
dye concentration on PCR and the eVect of dye concen-
tration on DNA melting curves. These data extend upon
the work of Giglio et al. [22] and demonstrate that,
unlike SYBR Green I, SYTO9 readily allows the detec-
tion of multiple amplicons in a multiplex PCR. The util-
ity of SYTO9 is demonstrated using both prokaryote
and eukaryote systems.
Materials and methods
Source of isolates and DNA template preparation
The source of V. cholerae O1 El Tor biotype and L.
pneumophila serotype 1 have been described previously
by Giglio et al. [22]. DNA was extracted from cultures
using Qiagen DNA-mini spin columns following the
manufacturer’s methods. The origins and characteriza-
tion of Giardia duodenalis isolates Ad-1, Ad-19, and Ad-
23, representative of Assemblages A, B, and F, respec-
tively, and of a Giardia ardeae isolate have been
described previously [24]. DNA samples from barley
(Horedum vulgare) varieties Atlas, Atlas68, Proctor, and
Shannon were kindly provided by Dr. Brendon King.
DNA concentrations for puriWed extracts were deter-
mined by measuring UV absorbance at 260nm.
Real-time PCR and melting curve analysis of prokaryotic
DNA
Real-time reactions were conducted in 100-?l thin-
walled tubes and monitored using a Rotor Gene 3000
(Corbett Research, Sydney, Australia). The multiplex
PCRs for V. cholerae and L. pneumophila were per-
formed as described previously [20], except that the V.
cholerae multiplex assay was modiWed to include DMSO
(5% v/v Wnal concentration), primer H3F was excluded,
and the primer concentrations of the remaining four
primers were reduced to 0.5?M each (Wnal concentra-
tion). The primer set 27f(5?-AGAGTTTGATYMTGG
CTCAG-3?) and 1492r (5?-TACGGYTACCTTGTT
ACGACT-3?), modiWed from Suzuki and Giovannoni
[25], was used to amplify a 1.46-kb fragment of the 16S
rRNA gene from V. cholerae DNA. Each 20-?l reaction
mixture contained 200?M each deoxynucleoside tri-
phosphate, 0.5?M each primer, 2.5mM MgCl2, 1£ PCR
buVer II (Applied Biosystems Foster City, CA, USA),
5%v/v DMSO, 1U of AmpliTaq Gold DNA polymerase
(Applied Biosystems), and 5?l of genomic DNA. Ther-
mal cycling consisted of an initial denaturation at 95°C
for 10min followed by 50 cycles of denaturation at 94°C
for 30s, annealing at 50°C for 1min, and extension at
72°C for 2min.
Dye concentrations of SYTO9 (Molecular Probes,
OR) or SYBR Green I used in reactions ranged from 10
to 0.5?M. The molar concentration of SYBR Green I
was calculated using the values determined by Zipper et
al. [26], who estimated that a 1£ SYBR Green 1 solution
represents a dye concentration of 2?M. Fluorescence

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Real-time PCR and melting curve analysis using SYTO9 / P.T. Monis et al. / Anal. Biochem. 340 (2005) 24–34
data were acquired on the FAM channel (excitation at
470nm, detection at 510nm) at the end of each 72°C
extension step of the PCR. The FAM channel closely
matches the excitation and emission maxima for SYTO9
(485 and 498nm, respectively, according to the manufac-
turer’s speciWcations). Fluorescence was measured over a
range of gain settings to accommodate the detection of
diVerent dye concentrations within a single experiment.
This was achieved using a feature of the RotorGene
3000 that allows the creation of multiple FAM channels,
each with diVerent gains ranging from 10 to ¡1.
Following ampliWcation,
acquired on the FAM channel using 1°C steps with a
hold of 60s at each step from 70 to 99°C. The diVerenti-
ated data were analyzed using the Rotor Gene software
(v6.0 build 14) with the digital Wlter set as “none.” When
required, samples were analyzed by 1% agarose gel elec-
trophoresis with the addition of Gelstar nucleic acid
stain (Cambrex Bio Science, Rockland, IN) using stan-
dard methods [27].
melting curves were
Determination of dye stability and between-run
reproducibility
Master mixes were prepared for the three bacterial
PCR assays described above and 20-?l aliquots were dis-
pensed into 100-?l tubes using a CAS-1200 liquid han-
dling system (Corbett Research). Each 20-?l aliquot
contained 25,000 or 25pg of DNA and either 2 or
0.5?M dye. Aliquots were stored in the dark at ¡20°C
until used. Real-time PCR was conducted on a Rotor-
Gene 3000 (Corbett Research) as described above. A
threshold value of 0.02 was used to determine the Ct
(cycle that crosses the threshold) value for each reaction.
For the multiplex reactions, the Ct values represent the
combined signal for both amplicons. This information
cannot be used for quantitation, but it does provide a
measure of assay reproducibility because it would be
expected that changes in the ampliWcation of either frag-
ment will aVect the total Xuorescence and therefore aVect
the Ct.
Real-time PCR and melting curve analysis of eukaryotic
DNA
The YLM locus was ampliWed from barley DNA
using the YLMF (5? CAGGAGCTGGTGAAATAGT
GCCT 3?) and YLMR (5? TTAAAGGGCTCCGT
GAAGC 3?) primers of Paltridge et al. [28]. PCRs were
conducted on a Rotor-Gene 3000 using the following
conditions: 95°C for 10min and 50 cycles of 94°C for
10s, 58°C for10s, and 72°C for 15s. The reaction mix-
ture (25?l) contained 100ng of genomic DNA, 1£ PCR
buVer II, 1.5mM MgCl2, 200?M each deoxynucleoside
triphosphate, 1U of AmpliTaq Gold DNA polymerase
(Applied Biosystems), 0.5?M each primer, and 3.3?M
SYTO9. DNA melting curve analysis was monitored
from 70 to 95°C in 1°C increments using a 30-s hold at
each step on the FAM channel. AmpliWcation products
were characterized by gel electrophoresis (as described
above) to conWrm ampliWcation of the expected sized
fragment.
A 660-bp fragment of the gene encoding glutamate
dehydrogenase was ampliWed from Giardia DNA using
primers GDHGENF (5? CGYGTNCCNTGGNTGG
AYGAYGCYGG 3?) and GDHGENR (5? AGYTT
CTCCTCGKTGAASCC 3?) and the following condi-
tions: 95°C for 5min and 55 cycles of 94°C for 30s,
55°C for 30s, and 72°C for 60s. The reaction mixture
(20?l) contained 2?l of DNA extract, 1£ PCR buVer
(20mM Tris–HCl (pH 8.4), 50mM KCl), 1.25mM
MgCl2, 200?M each deoxynucleoside, 1U of Platinum
Taq DNA polymerase (Invitrogen Life Technologies,
Carlsbad, CA USA), 0.25?M each primer, 5% DMSO,
and 3.3?M SYTO9. Data were acquired at 72°C on the
FAM channel. DNA melting curve analysis (using ver-
sion 5.0 build 60 of the Rotor-Gene software) was moni-
tored from 60 to 99°C in 1°C increments using a 10-s
hold at each step on the FAM channel. AmpliWcation
products were characterized by gel electrophoresis (as
described above) to conWrm ampliWcation of the
expected sized fragment.
Statistical analysis
Statistical analyses (two-sample t test for independent
samples) were conducted using Origin version 7 (Origin-
Lab, MA, USA).
Results and discussion
EVect of dye concentration on DNA melting curves
The available product information for the SYTO
family of dyes states that they are nucleic acid stains and
provides absorption and emission spectra for each dye in
the presence of DNA and RNA (http://www.
probes.com/media/pis/mp07572.pdf). However, no infor-
mation on dye speciWcity with regard to dsDNA or
ssDNA is provided. To investigate this for SYTO9,
DNA melting curve experiments were conducted using
genomic DNA ampliWed by the Legionella multiplex
PCR in the absence of any dye. A small volume of
SYTO9 was added post-PCR to provide a concentration
of 3.3?M, which was selected because it is the working
concentration used to stain live cells. The volume of dye
added was kept small to minimize any change to the
buVer or MgCl2 concentrations. For comparison, SYBR
Green I was added to replicate samples at the same
concentration. The detection of distinct melting peaks
(Fig. 1) demonstrates that SYTO9 is speciWc for dsDNA

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Real-time PCR and melting curve analysis using SYTO9 / P.T. Monis et al. / Anal. Biochem. 340 (2005) 24–34
27
and that it produces results comparable with SYBR
Green I, although the Tm values determined by each dye
diVered from each other by 2–3°C.
To examine the eVect of dye concentration on melting
curves, similar experiments using a broad range of con-
centrations (0.5–33?M) of either SYTO9 or SYBR
Green I and either Legionella multiplex PCR or Vibrio
multiplex PCR ampliWcation products were conducted.
Comparison of amplicon Tm versus dye concentration
for the mip, Legionella 16S rDNA, and hlyA amplicons
demonstrated that the Tm increases with increasing dye
concentration (Fig. 2), suggesting that the dyes have a
stabilizing eVect on the dsDNA. This eVect was much
more pronounced for SYBR Green I, where the rate of
increase in the Tm was higher than that for SYTO9 for
dye concentrations above 2?M. In the case of SYTO9
the Tm plateaued for concentrations of dye at or below
2?M, and the maximum diVerence in Tm between low
(62?M) and high (33?M) dye concentrations was 2°C
for the three amplicons analyzed. In contrast, the Tm val-
ues determined using SYBR Green I did not plateau
until the dye concentration was 0.5?M, and the diVer-
ence in Tm between the lowest and the highest dye con-
centrations was 7°C for hlyA, 10°C for mip, and >10°C
for 16S (the Tm for the latter being >99°C in the pres-
ence of 33?M dye). The observation that SYBR Green I
has a greater enhancement on Tm compared to SYTO9
under the same conditions suggests either that SYBR
Green I binds more tightly to dsDNA or that more
SYBR Green I molecules can bind per unit DNA. This
Wnding is supported by the recent work of Zipper et al.
[26], which suggests that SYBR Green I has a dual mode
of binding to dsDNA which is dependant on the dye/
base pair ratio. The eVect of the dye concentration on
the Tm was reXected in the shape of the melting peaks
(Fig. 3). For SYTO9, which had relatively little eVect on
Tm, the shape of the melting peaks was not aVected by
dye concentration, as illustrated for the mip and 16S
rDNA amplicons in Fig. 3. In the case of SYBR Green I,
both amplicons could be detected at high dye concentra-
tions but the peaks were broad and diYcult to interpret.
As the concentration of SYBR Green I decreased, the
melting peaks became sharper, with best peak resolution
obtained for concentrations below 2?M. At lower con-
centrations of the SYBR Green I, the peak for mip
became more diYcult to detect or could not be detected
(Fig. 3).
Comparison of SYTO9 with SYBR Green I for real-time
PCR and DNA melting curve analysis
The utility of SYTO9 was assessed by using it to mon-
itor DNA ampliWcation in real-time for the Legionella
multiplex, Vibrio multiplex, and 16S rDNA PCRs. These
assays amplify fragments of 114bp (mip) and 386bp
(Legionella 16S rDNA), 385bp (ctxAB) and 497bp
(hlyA), and approximately 1.5kb (Vibrio 16S rDNA),
respectively. The Vibrio 16S rDNA fragment would nor-
mally be considered too large for real-time PCR and rep-
resents a challenge for ampliWcation if any of the
conditions aVect the reaction eYciency. All reactions
were conducted in triplicate, using 250,000–25pg of
Fig. 1. DNA melting curve analysis of ampliWed mip and 16S rDNA
fragments using 3.3 ?M either SYTO9 or SYBR Green I that has been
added post-PCR.
Fig. 2. Comparison of dye concentration versus Tm for mip, Legionella
16S rDNA, and hlyA amplicons.

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Real-time PCR and melting curve analysis using SYTO9 / P.T. Monis et al. / Anal. Biochem. 340 (2005) 24–34
DNA per reaction. For comparison, PCRs were also
conducted using SYBR Green I. Based on the results of
the initial melting curve experiments, SYTO9 concentra-
tions of 10, 5, 2, and 0.5?M and SYBR Green I concen-
trations of 10, 2, 1, and 0.5?M were investigated. These
values represent concentrations across the range where
the dyes had diVerent eVects on melting temperature.
The results, summarized in Table 1, demonstrate that
SYTO9 can be used in PCR over a broader range of con-
centrations than can SYBR Green I. The Legionella mul-
tiplex PCR was inhibited by SYBR Green I at dye
concentrations of 10 and 2?M. The smaller mip frag-
ment was ampliWed at a SYBR Green I concentration of
1?M, and both fragments were ampliWed using 0.5?M
dye. The ampliWcation of mip and 16S rDNA using
0.5?M SYBR Green I was aVected by the amount of
starting template in the assay. When starting with
250,000 pg of template DNA only the mip fragment
ampliWed (Fig. 4). However, both fragments ampliWed
using 25,000 pg of template DNA, suggesting that the
mip amplicon preferentially ampliWes at higher template
concentrations. As the amount of starting template
decreased, the height of the 16S rDNA peak increased
and the mip peak height decreased. Using 25pg of tem-
plate DNA the peak heights for the two amplicons were
similar. These results are consistent with the Wndings of
Giglio et al. [22] and suggest that in the presence of lower
amounts of amplicon (expected for reactions starting
with less template DNA) both amplicons can be detected
by melting curve analysis, but at higher amounts of
amplicon the dye becomes limiting and the amplicon
with the higher Tm is preferentially detected. In contrast,
SYTO9 concentrations of 5, 2, and 0.5?M allowed the
ampliWcation of both fragments in the Legionella multi-
plex assay. All reactions using 10?M SYTO9 and reac-
tions with 5 and 2?M SYTO9 starting with 250,000 pg
of template DNA ampliWed only the mip fragment. This
suggests that high SYTO9 concentrations or a combina-
tion of dye concentration and high starting template
causes the preferential ampliWcation of the smaller mip
fragment.
The Vibrio multiplex PCR was less aVected by dye
concentration than was the Legionella muliplex PCR.
AmpliWcation was inhibited by SYBR Green I at a con-
centration of 10?M, but the assay worked for the lower
dye concentrations. Only the hlyA amplicon could be
detected by DNA melting curve analysis although both
fragments ampliWed (data not shown). This result is con-
sistent with the Wndings of Giglio et al. [22]. In the case of
SYTO9, both hlyA and ctxAB were ampliWed in the
Fig. 3. Comparison of DNA melting curves obtained using Legionella DNA ampliWed with mip and 16S rDNA primers and diVerent concentrations
of SYTO9 or SYBR Green I.