Mechanistic Approach to the Problem of Hybridization Efficiency in Fluorescent In Situ Hybridization

Abstract:

In fluorescent in situ hybridization (FISH), the efficiency of hybridization between the DNA probe and the rRNA has been related to the accessibility of the rRNA when ribosome content and cell permeability are not limiting. Published rRNA accessibility maps show that probe brightness is sensitive to the organism being hybridized and the exact location of the target site and, hence, it is highly unpredictable based on accessibility only. In this study, a model of FISH based on the thermodynamics of nucleic acid hybridization was developed. The model provides a mechanistic approach to calculate the affinity of the probe to the target site, which is defined as the overall Gibbs free energy change (Δ

Applied and Environmental Microbiology.2004:70(12):7126

Department of Civil and Environmental Engineering, University of Wisconsin—Madison, Madison,

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