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Comparison of SYTO9 and SYBR Green I for Real-Time Polymerase

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Comparison of SYTO9 and SYBR Green I for Real-Time Polymerase ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 340 (2005) 24–34 www.elsevier.com/locate/yabio 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 of dyes, including YO-PRO-1 [8], SYBR Green I [9,10], method in many diagnostic and research laboratories. BEBO [11], and LC Green [12], have been evaluated for The market for this technology continues to expand and use in real-time PCR applications. While probe-based there have been many probe-based systems developed to detection methods continue to be developed and meet this demand, including Taqman probes [1], molecu- improved, progress for intercalating dyes has been rela- lar beacons [2], FRET1 probes [3], Scorpions [4], and tively slow. iFRET [5]. An alternative to probe-based detection is the SYBR Green I has become the preferred choice for use of Xuorescent double-stranded DNA (dsDNA)-spe- real-time PCR because it is cost eVective compared to ciWc intercalating dyes. Such a dye, ethidium bromide, probe-based systems, allows the generic detection of was used in the Wrst description of real-time PCR by ampliWed DNA, and can be used to diVerentiate DNA Higuchi et al. [6,7]. Since then, a relatively small number by DNA melting curve analysis [9,10]. However, SYBR Green I also has disadvantages that limit its ease of use. V ¤ It cannot generally be used in standard PCR bu ers Corresponding author. Fax: +61 8 8259 0228. without further optimization of conditions and the E-mail address: [email protected] (P.T. Monis). 1 Abbreviations used: FRET, Xuorescence resonance energy transfer; inclusion of additional reagents to improve reaction DMSO, dimethyl sulfoxide. eYciency, such as DMSO [13], bovine serum albumin, 0003-2697/$ - see front matter. Crown copyright 2005 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.01.046 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- unlike SYBR Green I, SYTO9 readily allows the detec- tions, the dye also appears to be inhibitory to PCR in a tion of multiple amplicons in a multiplex PCR. The util- concentration-dependent manner [9,15] which can be ity of SYTO9 is demonstrated using both prokaryote overcome by increasing the concentration of MgCl2 in and eukaryote systems. 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 Materials and methods widely applied for the identiWcation of a variety of organisms (e.g., Leptonema [16], Borellia [17,18], Legion- Source of isolates and DNA template preparation ella [19], and Cryptosporidium [20]). However, the melt- ing temperature (Tm) obtained using this dye can be The source of V. cholerae O1 El Tor biotype and L. aVected by the concentration of dye [10] and the concen- pneumophila serotype 1 have been described previously tration of DNA [21]. For example, in the study of Ririe by Giglio et al. [22]. DNA was extracted from cultures et al. [10] a 3 log-fold diVerence in the number of starting using Qiagen DNA-mini spin columns following the copies of target DNA used in real-time PCR resulted in manufacturer’s methods. The origins and characteriza- V a Tm di erence of more than 1 °C for the same amplicon. tion of Giardia duodenalis isolates Ad-1, Ad-19, and Ad- A further limitation of SYBR Green I is that it 23, representative of Assemblages A, B, and F, respec- appears to have limited application for the analysis of tively, and of a Giardia ardeae isolate have been multiplex PCR. In a study by Giglio et al. [22], multiplex described previously [24]. DNA samples from barley PCRs for Vibrio cholerae and Legionella pneumophila (Horedum vulgare) varieties Atlas, Atlas68, Proctor, and were analyzed by DNA melting curve analysis using Shannon were kindly provided by Dr. Brendon King. SYBR Green I. It was found that only one amplicon DNA concentrations for puriWed extracts were deter- could be detected by melting curve analysis but that mined by measuring UV absorbance at 260 nm. both amplicons were being ampliWed as determined by agarose gel electrophoresis. This eVect appeared to be Real-time PCR and melting curve analysis of prokaryotic related to the relative concentrations of the ampliWed DNA DNA and dye because time-based analysis demon- strated that both amplicons could be detected by melting Real-time reactions were conducted in 100-l thin- curve analysis after 20 cycles of PCR but only one walled tubes and monitored using a Rotor Gene 3000 amplicon could be detected after 40 cycles of PCR. (Corbett Research, Sydney, Australia). The multiplex The SYTO family of dyes have many properties that PCRs for V. cholerae and L. pneumophila were per- make them useful for labeling live cells, including good formed as described previously [20], except that the V. membrane permeability, low cytotoxicity, and enhanced cholerae multiplex assay was modiWed to include DMSO Xuorescence in the presence of DNA (US patent (5% v/v Wnal concentration), primer H3F was excluded, 5,436,134 [23]). Although they are ostensibly DNA-bind- and the primer concentrations of the remaining four ing dyes, their low toxicity suggests that they may not primers were reduced to 0.5 M each (Wnal concentra- interfere with cellular processes, such as DNA and RNA tion). The primer set 27f(5Ј-AGAGTTTGATYMTGG synthesis, at the concentrations that are used for staining CTCAG-3Ј) and 1492r (5Ј-TACGGYTACCTTGTT cells. Little information with regard to their speciWcity ACGACT-3Ј), modiWed from Suzuki and Giovannoni for dsDNA versus single-stranded DNA (ssDNA) or [25], was used to amplify a 1.46-kb fragment of the 16S their suitability as dyes for real-time PCR is available. rRNA gene from V. cholerae DNA. Each 20-l reaction Evaluation of the mode of action of the BacLight Live/ mixture contained 200 M each deoxynucleoside tri- Dead kit (Molecular Probes) in our laboratory suggested phosphate, 0.5 M each primer, 2.5 mM MgCl2, 1£ PCR that SYTO9 is a dsDNA-speciWc dye. These factors, buVer II (Applied Biosystems Foster City, CA, USA), together with the similarity in excitation/emission max- 5% v/v DMSO, 1 U of AmpliTaq Gold DNA polymerase ima in SYTO9 and the FAM channel of the Rotor Gene (Applied Biosystems), and 5 l of genomic DNA. Ther- 3000, have led us to select SYTO9 for evaluation in real- mal cycling consisted of an initial denaturation at 95 °C time PCR. for 10 min followed by 50 cycles of denaturation at 94 °C We report herein the novel use of SYTO9 in real-time for 30 s, annealing at 50 °C for 1 min, and extension at PCR. This dye supports PCR ampliWcation over a wide 72 °C for 2 min. range of dye concentrations and produces robust DNA Dye concentrations of SYTO9 (Molecular Probes, melting curves that are not aVected by DNA concentra- OR) or SYBR Green I used in reactions ranged from 10 tion. In addition, we investigated the inhibitory eVect of to 0.5 M. The molar concentration of SYBR Green I dye concentration on PCR and the eVect of dye concen- was calculated using the values determined by Zipper et tration on DNA melting curves. These data extend upon al. [26], who estimated that a 1£ SYBR Green 1 solution the work of Giglio et al. [22] and demonstrate that, represents a dye concentration of 2 M. Fluorescence 26 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 SYTO9. DNA melting curve analysis was monitored 470 nm, detection at 510 nm) at the end of each 72 °C from 70 to 95 °C in 1 °C increments using a 30-s hold at extension step of the PCR. The FAM channel closely each step on the FAM channel.
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