Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models

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.

• DNA
• locked nucleic acids
• nucleic acid mimics
• peptide nucleic acids

Declaration of interest

This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE, ON.2 – O Novo Norte – North Portugal Regional Operational Programme and National Funds through FCT – Foundation for Science and Technology under the projects: PEst-C/EQB/UI0511, NORTE-07-0124-FEDER-000025 – RL2_ Environment&Health and DNA mimics Research Project PIC/IC/82815/2007, PhD Fellowship SFRH/BD/72999/2010 and Post-Doctoral fellowship SFRH/BPD/78846/2011. The Nucleic Acid Center, University of Southern Denmark is thanked for financial support.

JW is cofounder of RiboTask ApS which offer LNA/2′-OMe-RNA probes for RNA targeting. NFA is cofounder of Biomode SA which develops molecular methods for the rapid detection of microorganisms.

Supplementary material available online