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Biopolym. Cell. 2025; 41(4):300.
http://dx.doi.org/10.7124/bc.000B2C
Bioorganic Chemistry
Evaluation of the monomethine cyanine dye FB128 for real-time PCR applications
1Kazakov-Kravchenko O. S., 1, 2Kravchenko S. A., 1Yarmoluk S. M.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
  2. Kyiv Institute of the National Guard of Ukraine, MIA of Ukraine
    7, Oborony Kyieva Str., Kyiv, Ukraine, 03179

Abstract

Aim. To evaluate the suitability of the novel DNA-binding dye fb128 for real-time PCR applications, including amplification efficiency, fluorescence performance, PCR inhibition threshold, and melting curve analysis. Methods. UV-vis absorption and fluorescence spectroscopy, agarose gel electrophoresis, real-time PCR, melt curve analysis. Results. fb128 exhibited low intrinsic fluorescence and strong DNA-specific signal (ΔQ = 160.7). Optimal concentrations (0.2—1.6 μM) provided early Ct values without PCR inhibition, whereas the concentrations of ≥3 μM caused a delayin amplification or complete suppression. PCR efficiency with fb128 was 102.6%, within the optimal range for quantitative applications. Melting peaks with fb128 were stronger and appeared at 74.5 °C, 2.5 °C lower than with SYBR Green I, suggesting weaker dsDNA binding. Conclusions. fb128 demonstrates high amplification efficiency, strong fluorescent signal, broad concentration tolerance, and robust melting analysis performance. These properties establish fb128 as a competitive alternative to SYBR Green I for real-time PCR applications.
Keywords: monomethine cyanine dye, fluorescence intensity, real-time PCR, DNA binding, PCR efficiency, melt curve analysis
Full text: (PDF, in English)

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