Biopolymers, 2011, Vol. 95, No. 7, pp 472-486.
Measuring thermodynamic details of DNA hybridization using fluorescence
Yong You, Andrey V. Tataurov, and Richard Owczarzy
Modern real-time PCR systems make it easy to monitor fluorescence while
temperature is varied for hundreds of samples in parallel, permitting
high-throughput studies. We employed such system to investigate melting
transitions of ordered nucleic acid structures into disordered random coils.
Fluorescent dye and quencher were attached to oligonucleotides such a way that
changes of fluorescence intensity with temperature indicated progression of
denaturation. When fluorescence melting data were compared with traditional
ultraviolet optical experiments, commonly used dye/quencher combinations,
like fluorescein and tetramethylrhodamine, showed substantial discrepancies.
We have therefore screened 22 commercially available fluorophores and
quenchers for their ability to reliably report annealing and melting
transitions. Dependence of fluorescence on temperature and pH was also
investigated. The optimal performance was observed using Texas Red or ROX dyes
with Iowa Black RQ or Black Hole quenchers. These labels did not alter
two-state nature of duplex melting process and provided accurate melting
temperatures, free energies, enthalpies and entropies. We also suggest a new
strategy for determination of DNA duplex thermodynamics where concentration
of a dye-labeled strand is kept constant and its complementary strand
modified with a quencher is added at increasing excess. These methodological
improvements will help build predictive models of nucleic acid hybridization.
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