Biochemical and Biophysical Research Communications, 2005, Vol. 327, No. 2, pp 473-484.
Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability
Bernardo G. Moreira, Yong You, Mark A. Behlke, and Richard Owczarzy
Reprint
Supplementary data
Single and dual-labeled fluorescent oligodeoxynucleotides are used in many
molecular biology applications. We investigated the effects of commonly used
fluorescent dyes and quenchers on the thermodynamic stability of a model
probe-target DNA duplex. We demonstrate that those effects can be significant.
Fluorescent dyes and quenchers were attached to the probe ends. In certain
combinations, these groups stabilized the duplex up to 1.8 kcal/mol and
increased Tm up to 4.3 oC. None of the groups tested significantly
destabilized the duplex. Rank order of potency was, starting with the most
stabilizing group: Iowa Black RQ ~ Black Hole 2 > Cy5 ~ Cy3 > Black Hole 1 >
QSY7 ~ Iowa Black FQ > Texas Red ~ TAMRA > FAM ~ HEX ~ Dabcyl > TET. Longer
linkers decreased stabilizing effects. Hybridizations to targets with various
dangling ends were also studied and were found to have only minor effects on
thermodynamic stability. Depending on the dye/quencher combination employed,
it can be important to include thermodynamic contributions from fluorophore
and quencher when designing oligonucleotide probe assays.
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