Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications

Background: EvaGreen (EG) is a newly developed DNA-binding dye that has recently been used in quantitative real-time PCR (qPCR), post-PCR DNA melt curve analysis and several other applications. However, very little is known about the physicochemical properties of the dye and their relevance to the applications, particularly to qPCR and post PCR DNA melt curve analysis. In this paper, we characterized EG along with a widely used qPCR dye, SYBR Green I (SG), for their DNA-binding properties and stability, and compared their performance in qPCR under a variety of conditions. Results: This study systematically compared the DNA binding profiles of the two dyes under different conditions and had these findings: a) EG had a lower binding affinity for both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) than SG; b) EG showed no apparent preference for either GC- or AT-rich sequence while SG had a slight preference for AT-rich sequence; c) both dyes showed substantially lower affinity toward ssDNA than toward dsDNA and even lower affinity toward shorter ssDNA fragments except that this trend was more pronounced for EG. Our results also demonstrated that EG was stable both under PCR condition and during routine storage and handling. In the comparative qPCR study, both EG and SG exhibited PCR interference when used at high dye concentration, as evident from delayed Ct and/or nonspecific product formation. The problem worsened when the chain extension time was shortened or when the amplicon size was relatively long (> 500 bp). However, qPCR using EG tolerated a significantly higher dye concentration, thus permitting a more robust PCR signal as well as a sharper and stronger DNA melt peak. These differences in qPCR performance between the two dyes are believed to be attributable to their differences in DNA binding profiles. Conclusion: These findings suggest that an ideal qPCR dye should possess several DNA-binding characteristics, including a "just right" affinity for dsDNA and low or no affinity for ssDNA and short DNA fragments. The favorable DNA-binding profile of EG, coupled with its good stability and instrument-compatibility, should make EG a promising dye for qPCR and related applications.