Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature

Abstract

The importance of real-time polymerase chain reaction (PCR) has increased steadily in clinical applications over the last decade. Many applications utilize SYBR Green I dye to follow the accumulation of amplicons in real time. SYBR Green I has, however, a number of limitations that include the inhibition of PCR, preferential binding to GC-rich sequences and effects on melting curve analysis. Although a few alternative dyes without some of these limitations have been recently proposed, no large-scale investigation into the properties of intercalating dyes has been performed. In this study, we investigate 15 different intercalating DNA dyes for their inhibitory effects on PCR, effects on DNA melting temperature and possible preferential binding to GC-rich sequences. Our results demonstrated that in contrast to the results of SYBR Green I, two intercalating dyes SYTO-13 and SYTO-82 do not inhibit PCR, show no preferential binding to GC rich sequences and do not influence melting temperature, T(m), even at high concentrations. In addition, SYTO-82 demonstrated a 50-fold lower detection limit in a dilution series assay. In conclusion, the properties of SYTO-82 and SYTO-13 will simplify the development of multiplex assays and increase the sensitivity of real-time PCR.

Figure 1.

Comparison of the threshold cycle (C_t) value obtained for six example dyes using the UB PCR to demonstrate the different characteristics of the dyes. The PCR inhibition factor was defined as the average slope for all three PCR products. (See also Supplementary Figure S1)

Figure 2.

Comparison of dye concentration versus T_m of the HIP amplicon for all four optical channels (A, B, C and D).

Figure 3.

Example of multiplex melting curve separation of two products with different concentrations of SYTO-82 (A and B) and SYBR Green I (C and D). The melting curves were for the pre-amplified multiplex PCR with both UC (144 bp, GC 51%, right peak) and HIP (149 bp, GC 37%, left peak) primers. The height of the peak was calculated as the peak height from a baseline defined by the background and the height peak ratio is the ratio of the right peak divided by the left.

Figure 4.

Comparison of dye concentration versus the peak height ratio for a few selected dyes in the UC/HIP multiplex PCR reactions (For other dyes see also Supplementary Data Figure S3). The degree of preferential binding for the dyes (low, medium, or high) is indicated in the figure.

Figure 5.

The real-time PCR detection limit when using SYBR Green I and SYTO-82 was evaluated by the melting curve peak area. The HIP PCR was performed with a large gradient of template DNA ranging from 10⁶ to 50 copies of the C. jejuni DNA. For SYBR Green I the PCR product melting peak area was measured for both the dye concentrations of 2 and 0.2 μM.