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Applied and Environmental Microbiology, December 2004, p. 7126-7139, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7126-7139.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

L. Safak Yilmaz and Daniel R. Noguera*

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

Received 25 March 2004/ Accepted 22 July 2004

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 ({Delta}G°overall) for a reaction scheme involving the DNA-rRNA and intramolecular DNA and rRNA interactions that take place during FISH. Probe data sets for the published accessibility maps and experiments targeting localized regions in the 16S rRNA of Escherichia coli were used to demonstrate that {Delta}G°overall is a strong predictor of hybridization efficiency and superior to conventional estimates based on the dissociation temperature of the DNA/rRNA duplex. The use of the proposed model also allowed the development of mechanistic approaches to increase probe brightness, even in seemingly inaccessible regions of the 16S rRNA. Finally, a threshold {Delta}G°overall of –13.0 kcal/mol was proposed as a goal in the design of FISH probes to maximize hybridization efficiency without compromising specificity.

* Corresponding author. Mailing address: Dept. Civil and Environmental Engineering, University of Wisconsin, 1415 Engineering Dr., Madison, WI 53706. Phone: (608) 263-7783. Fax: (608) 262-5199. E-mail: noguera{at}engr.wisc.edu.

Applied and Environmental Microbiology, December 2004, p. 7126-7139, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7126-7139.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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