Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models |
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| Authors: | Sílvia Fontenete Nuno Guimarães Jesper Wengel |
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| Affiliation: | 1. Department of Chemical Engineering, Faculty of Engineering, LEPABE, University of Porto, Porto, Portugal,;2. Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal,;3. Department of Physics, Chemistry and Pharmacy, Nucleic Acid Center, University of Southern Denmark, Odense M, Denmark, and;4. ICBAS, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal;5. Department of Physics, Chemistry and Pharmacy, Nucleic Acid Center, University of Southern Denmark, Odense M, Denmark, and;6. Department of Physics, Chemistry and Pharmacy, Nucleic Acid Center, University of Southern Denmark, Odense M, Denmark, and |
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| Abstract: | ![]()
The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescence in situ hybridization (FISH) procedure, the melting temperature is used as a reference, together with corrections for certain compounds that are used during FISH. However, the quantitative relation between melting and experimental FISH temperatures is poorly described. In this review, various models used to predict the melting temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically modified oligonucleotides), will be addressed in detail, together with a critical assessment of how this information should be used in FISH. |
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| Keywords: | DNA locked nucleic acids nucleic acid mimics peptide nucleic acids |
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