New unsymmetrical cyanine dyes for real-time thermal cycling

Asymmetric cyanine dyes bind to the minor groove of double stranded DNA (dsDNA) owing to their crescent configuration; therefore, these dyes are widely used as a dsDNA probes. BOXTO-MEE is derived from BOXTO by adding the polar methoxyethoxyethyl tail in order to increase solubility, dissociation rate kinetics, and stability. As a result, BOXTO-MEE showed significant reduction in nonspecific amplification (primer dimers) without significant effect on target sequence amplification, PCR efficiency, and standard curve correlation coefficient. BETIBO is another example of an asymmetric cyanine dye that can binds to dsDNA but is less efficient than BOXTO-MEE for use in real-time PCR. Statistical analysis of reproducibility results shows that BETIBO is not strong enough to be used for quantifying low nucleic acid quantities. Statistical analysis for BOXTO-MEE results shows that there is no significant difference between the efficiency and correlation coefficient achieved by BOXTO-MEE and SYBR Green I, but a significant difference in the dynamic range is observed because BOXTO-MEE has a wider dynamic range. BOXTO-MEE stock solution was stable at −20 °C for more than 1 year and 40 μM solution was stable for 45 days (at least) at 4 °C.