Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes

Since their introduction some three decades ago, methods for hybridization analysis of nucleic acids immobilized on solid supports have evolved to imp rove the sensitivity, speed, and convenience of their application. However, in many cases these methods still require the use of solutions containing formamide, a recognized hazardous solvent with potential toxicity. Here, we have compared the efficiency of urea to that of formamide as denaturing ag ent in nucleic acid hybridization with RNA probes. We show that urea at con centrations of 2-4 molar in solution performs as good as 50% formamide to r educe heterologous background hybridization in Northern blotting experiment s realized at 68 degreesC. Presence of urea at higher concentrations result ed in reduced hybridization sensitivity, possibly due to increased viscosit y. When tested in Southern blot analysis of genomic DNA, our results reveal ed that the use of urea in hybridization solution is also suitable to carry out single-copy gene detection. Together, these findings show that urea ca n efficiently and safely replace formamide in solutions.