Alexander Zielske with Dr. Daniel Noguera and Dr. Safak Yilmaz

Department of Civil and Environmental Engineering, UW-Madison

Fluorescent In Situ Hybridization, or “FISH”, is a molecular technique used to quickly analyze specific microorganisms in a microbial community. Using the FISH technique, fluorescent DNA probes were hybridized with rRNA target sites with the hope of better understanding the effects of DNA/rRNA mismatches on probe affinity. The probe affinity is defined as the overall free energy change (∆G0overall) of hybridization, which is based on Gibbs Free Energy for thermodynamics. In relation to the probe affinity, the DNA probe’s location and its mismatch type were investigated specifically.

For this study, three fluorescent DNA probes were designed with different probe/target mismatches, and then hybridized with specific sites on 16S rRNA in E. coli at varying formamide percentages. The hybridized DNA probes were analyzed using flow cytometry with the results represented in formamide dissociation curves. Using the dissociation curves and other literature, both the predicted probe affinity and actual probe affinity can be evaluated in relation to the given mismatch type, and the location of the mismatch on the target rRNA site.

By using the results of such research, it is hoped that the impact of single duplex mismatches on probe affinity in the FISH technique can be better understood, with the ultimate goal being an improved FISH methodology.