We have investigated a panel of fluorescent dyes for use in real-time quantitative PCR. The dye SYTO-82 performed better than any of the other dyes tested. It did not preferentially bind to GC- or AT-rich sequences, did not inhibit PCR significantly and demonstrated a 50-fold lower detection limit in a real-time PCR assay compared to SYBR Green I. In contrast, SYBR Green I showed preferential binding to GC-rich sequences, a phenomena previously demonstrated by Giglo et al.
]. At a concentration of 2 μM, SYBR Green I inhibited each PCR cycle by about 22% while at a concentration of 20 μM SYBR Green I completely inhibited PCR. Such dose-dependent inhibition was not observed for SYTO-82. The decreased detection limit in real-time PCR with SYTO-82 compared to SYBR Green I can be explained by the higher PCR inhibition of SYBR Green I.
The melting temperature of the PCR products was always at least 2°C over the calculated melting temperature. The systematic overestimation of the measured melting temperature may be caused by the dyes binding to the DNA. With the exception of SYTO-13, SYTO-64 and SYTO-82, all other dyes showed a significant increase of melting temperature of the DNA hybrid with increasing dye concentration. This suggests that there is a common stabilizing effect of the dyes on the DNA double helix. The effect was mostly observed at dye concentrations above 2 μM. For instance, SYBR Green I, SYTOX Orange and YO-PRO-1 increased the measured Tm
between 6° and 10°C at a 20 μM dye concentration. A study performed by Monis et al.
] that compares SYBR Green I and SYTO-9 in a similar manner, revealed a Tm
difference of 10°C for 20 μM SYBR Green I. This result is in agreement with our results. There could also be a machine-dependent cause for melting curve temperature differences as suggested by Herrman et al.
]. This difference in melting curves of real-time PCR using SYBR Green I was reported to be in the order of 0.5°C between instruments. This difference is however, negligible when compared with the effect of high concentration of dyes (10°C). The melting temperature shift phenomenon correlates very closely to the measured inhibition effect when both the inhibition, as represented by the Ct
value slope, and Tm
shift are compared together as an average for all three PCR amplicons. The dyes demonstrate a linear correlation suggesting a close relationship between the inhibition of PCR and Tm
shift (Supplementary Data Figure S5). This indicates that the tightness of the dye binding could influence the annealing of the primers or possibly alter the ability of the polymerase to function optimally. This correlation should make it possible to predict, based on the Tm
shift alone, how significant the inhibition effect is for a specific intercalating dye. The PCR inhibition caused by the dyes has a negative effect on the sensitivity of any given assay as could be seen in the comparison between SYBR Green I and SYTO-82, and therefore the use of SYTO-82 could enhance existing real-time detection assays by increasing PCR efficiency.
The preferential binding of SYBR Green I to specific DNA fragments containing higher GC% content and larger sizes in multiplex PCR with low dye concentrations was demonstrated by Giglio et al.
]. We confirmed this phenomenon for SYBR Green I and other dyes. The preferential binding of the dyes, as measured by the peak ratio, was similarly compared against the Tm
shift demonstrating a linear relationship between the preferential binding of the dye and the Tm
shift. (Supplementary Data Figure S6) This suggests a close connection between the DNA affinity of the dyes and the three properties: amplification inhibition, Tm
shift and preferential binding.
Information from the literature [32–35
] and from the manufacturer indicates that inhibition of amplification, Tm
shift and preferential binding can be explained by the affinity of the dye to dsDNA. According to US patent US 5658751, the structure of SYBR Green I is based on a monomeric unsymmetrical cyanine dye [28
] with high DNA binding affinity. The TOTO dyes are symmetric dimeric nucleic acid stains that are among the most sensitive and highest affinity fluorescent dyes available for nucleic acid staining while its relative, the TO-PRO family, are smaller monomeric asymmetric cyanine dyes with lower DNA affinity. SYTOX Orange is classified as a SYTO dye but according to Molecular Probes (Invitrogen, CA, USA) it is a high affinity nucleic acid dye. The SYTO dyes are cell permeable cyanine dyes with a relatively low-affinity for nucleic acids [32
]. Comparison of the overall performance of the dye families to their reported DNA affinity suggests that DNA affinity is a defining factor for the performance of the dyes as high affinity dyes inhibits PCR to a higher degree compared to low affinity dyes. This can be seen with low affinity SYTO dyes that have the best overall performance while high affinity dyes such as SYBR Green I and SYTOX Orange have very poor performance. In addition, the very high affinity TOTO dyes demonstrate complete PCR inhibition, suggesting that this dye family may interfere with the performance of the polymerase or even primer binding. These results confirm that the tightness of the binding of the dye to DNA influences the measured Tm
, PCR product preference and PCR inhibition.
Following an extensive screening of fluorescent intercalating DNA dyes we have concluded that there are a number of dyes suitable for real-time PCR. Among the 14 dyes tested, most notable are the dyes SYTO-13 and SYTO-82 which performed superbly on all aspects tested such as having a minimal inhibitory effect on PCR, minimal preferential binding and minimal melting temperature shift. The cost difference between SYTO-82 and SYBR Green I is about 7-fold. However, the cost of the dye is not an important factor when compared to the overall cost of the PCR reaction. In our case, the cost of Amplitaq Gold is about 93 cents (US)/reaction and that corresponds to a dye cost of 0.1% of the total reagent cost for SYBR Green I and 0.6% for SYTO-82. Furthermore, the detection limit using SYTO-82 in a real-time PCR application was 50-fold better than SYBR Green I that easily compensates for the slightly higher cost of SYTO-82. In conclusion, our results strongly suggest that SYTO-13 and SYTO-82 should replace SYBR Green I in real-time PCR applications.