Blending DNA binding dyes to improve detection in real-time PCR

- Humic acid quenches the fluorescence of the dsDNA binding dyes EvaGreen, ResoLight, SYBR Green I and SYTO9.
- A four-dye blend containing the dyes above provides maximum fluorescence signals in presence and absence of humic acid.
- Blending complementary dyes enables improved qualitative detection of DNA in samples with high amounts of humic acid.
- Humic acid interactions with dsDNA binding dyes lower the Tm of amplicons, thereby disturbing melt curve analysis.

The success of real-time PCR (qPCR) analysis is partly limited by the presence of inhibitory compounds in the nucleic acid samples. For example, humic acid (HA) from soil and aqueous sediment interferes with amplification and also quenches the fluorescence of double-stranded (ds) DNA binding dyes, thus hindering amplicon detection. We aimed to counteract the HA fluorescence quenching effect by blending complementary dsDNA binding dyes, thereby elevating the dye saturation levels and increasing the fluorescence signals. A blend of the four dyes EvaGreen, ResoLight, SYBR Green and SYTO9 gave significantly higher fluorescence intensities in the presence and absence of HA, compared with the dyes applied separately and two-dye blends. We propose blending of dyes as a generally applicable means for elevating qPCR fluorescence signals and thus enabling detection in the presence of quenching substances.