New methods for detection of specific DNA segments using fluorescence energy transfer probes allow DNA amplification and detection to be performed in a closed system, which has great promise for clinical applications of DNA diagnostics. The sensitivity of such assays is usually about 100-1000 target DNA molecules in a 10 microliter reaction. We found that by reducing the volume of such reactions 100- to 1000-fold we could routinely detect single DNA target molecules. This is important because it makes it feasible to quantitate the number of starting DNA molecules by performing replicate reactions on limiting dilutions of sample and counting the number of positive reactions. To facilitate this kind of analysis, we developed a microscope slide-like device that automatically partitions a ~ 10 microliter drop of reaction solution into ~ 100 subportions, each of which is protected from evaporation during the DNA amplification. The partitioning mechanism relies on surface tension forces. Results of amplification in the slide-like device were readable with flat plate fluorescence imagers commonly used for analysis of DNA gels and membranes. Limiting dilution of genomic DNA indicated that the array devices detected single copies of target DNA/well. We developed a model of the droplet formation process using computer simulation and other mathematical methods. The model predicts conditions in terms of hydrophobicity of materials and device dimensions and geometry for droplets to form. Development of the slide-like device is being done in collaboration with an industrial partner with expertise in surface coatings, under a cooperative research and development agreement.
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