Abstract
BackgroundReal-time PCR can be carried out using either probes or DNA dyes. SYBR Green has been used the most, but it suffers from several drawbacks. Numerous other DNA dyes are commercially available, but with limited structural information. Dye behavior in real time PCR is difficult to predict, so empirical data are needed. In the work described here, a panel of 23 different DNA dyes--including green, orange, and red SYTO dyes, EvaGreen, and SYBR Green--were evaluated with respect to their performance in real time PCR.FindingsData were analyzed for reaction inhibition, effects on amplicon melting temperature, fluorescent signal strength, and reaction efficiency. This is the first report of reaction efficiency using alternatives to SYBR Green. Results indicated substantial variation in performance even within the SYTO dye family. EvaGreen and the SYTO dyes 13, 16, 80, and 82 performed better than SYBR Green in general, and high reaction efficiencies can be achieved using these dyes.ConclusionsEmpirical data were generated for 23 DNA dyes. This paper confirms and extends previous findings that among commercially available DNA dyes, EvaGreen and certain SYTO dyes are the most desirable alternatives to the commonly used SYBR Green in real-time PCR.
Highlights
Real-time PCR can be carried out using either probes or DNA dyes
Empirical data were generated for 23 DNA dyes
Real-time PCR can be carried out using sequence-specific oligonucleotide probes or non-sequence specific DNA dyes [1,2]
Summary
Real-time PCR can be carried out using either probes or DNA dyes. In the work described here, a panel of 23 different DNA dyes–including green, orange, and red SYTO dyes, EvaGreen, and SYBR Green–were evaluated with respect to their performance in real time PCR. Real-time PCR can be carried out using sequence-specific oligonucleotide probes or non-sequence specific DNA dyes [1,2]. Dyes offer greater flexibility and reduced cost, and they allow dissociation (melt) curve analysis of PCR products, while the most commonly used probes do not [2,3,4]. Despite the advantages of dye-based detection and the wide variety of commercially available fluorescent DNA dyes, most real-time PCR has been conducted using SYBR Green. Melt curve analysis using SYBR Green is complicated by the dye’s effect on melting temperature and by
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