Measuring thermodynamic details of DNA hybridization using fluorescence

Abstract

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.

DNA duplex stabilization in crowded polyanion solutions

“…1,2 15 While most biochemical reactions have been studied only in simple buffers, the concept of macromolecular crowding has been increasingly appreciated in the past few decades.…”

“…DNA melting was thus monitored by fluorescence enhancement. [15][16][17][18][19] The melting curves in the presence of increasing concentrations of NaPAA1200 are shown in Figure 2A, where typical DNA melting transitions are observed. The temperature corresponding to the maximal of the first 65 derivative of a melting curve is Tm.…”

Amplifying the Macromolecular Crowding Effect Using Nanoparticles

“…This fluorescence method was previously reported, 14 and a comparison to the tradition method based on the UV hyperchromic effect has been recently carried out. 15 Based on this report, our fluorescent DNA should give precise and accurate T m results.The melting curves of the AuNP-free fluorescent DNA in water, 5% (w/w) glycerol, ethylene glycol (EG), or PEGs of different MWs are shown in Figure 2A. All the solutions also contained 50 mM NaCl, 10 mM HEPES, pH 7.6.…”

“…This fluorescence method was previously reported, 14 and a comparison to the tradition method based on the UV hyperchromic effect has been recently carried out. 15 Based on this report, our fluorescent DNA should give precise and accurate T m results.…”

Microheater platform for selective detachment of DNA

“…Moreover, fluorescence of Texas Red is a very weak function of temperature in the temperature range of interest. 17 It is expected that this work may enable the development of DNA analysis microdevices wherein sophisticated temperature zones are defined to enable DNA to be detached only from desired locations in a glass-based DNA analysis chip. Such an exercise may utilize thermal simulations discussed previously to reverse-engineer the microheater line pattern required to produce a desired temperature map.…”