Solving the Problem of Specificity in Fluorescence In Situ Hybridization

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Fluorescence in situ hybridization (FISH) is an integral part of the full-cycle rRNA approach to the identification and quantification of microorganisms in a community. This technology has already demonstrated useful in many environmental engineering applications such as activated sludge, anaerobic digesters, biofilms, and natural environments. Due to its increasing importance, the research presented in this paper is focused on improving efficiency for users of FISH. In particular, the goal was to gain predictive power over the performance of probes regarding specificity in FISH by producing a ranking of mismatches through incorporating three key variables: mismatch type, mismatch position, and nearest neighbors. In the end, the mismatch stability ranking determined was based on the first two key variables: (most stable) rUdG < rGdT ≈ rUdT < rUdC < rAdG < rGdG ≈ rAdA ≈ rAdC < rGdA (least stable). The third variable, nearest neighbors, was addressed using the theoretically determined ΔΔG°1. This proved to be well correlated (R2 value of 0.6097) with the experimental results, indicating that a theoretical base for predictability is possible.
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