Formamide-Free Fluorescence In Situ Hybridization (FISH)

Abstract

Formamide is an ionising solvent which is widely used in molecular biology research for its thermodynamic effects on the DNA double-helix stability. In fluorescence in situ hybridization (FISH), the addition of formamide to aqueous buffers solutions of DNA enables key procedural steps—such as the prehybridization denaturation, the reannealing step and the post-hybridization stringency washes—to be carried out at lower, less harsh temperatures without compromising the overall efficiency and specificity of the hybridization. However, formamide is toxic and a potential teratogen, and its use in research laboratories demands implementation of specific safety measures and the introduction of precautionary steps which can potentially complicate the logistical flow. This chapter outlines two simple, effective alternatives to the standard FISH protocol in which the use of formamide in sizable volumes is essentially eliminated and replaced by the utilisation of ‘safer’ chemicals. These developments might be of particular interest to research scientists considering the implementation in their laboratories of safety solutions and long-term sustainability strategies aimed at simplifying procedures, reducing exposure risks for the workers and minimising production of toxic waste.