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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 in 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.

SUBMITTER: You Y

PROVIDER: S-EPMC3082624 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Live-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell's integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique. Results:Presented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm's accurate assessment of DNA content was validated by parallel flow cytometric studies. Conclusions:This method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.

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Measuring thermodynamic details of DNA hybridization using fluorescence.

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